Growth Hormone-Releasing Hormone Antagonists and Uses Thereof

ABSTRACT

Described herein are compositions and methods for treating pulmonary fibrosis and cancer. The compositions include growth hormone releasing hormone peptides. The methods include reducing lung inflammation, lung scarring, reducing expression of T cell receptor complex genes as well as inhibiting tumor growth.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/803,170, filed Feb. 8, 2019, and U.S. Provisional Application No.62/869,687, filed Jul. 2, 2019. The content of these earlier filedapplications is hereby incorporated by reference herein in its entirety.

INCORPORATION OF THE SEQUENCE LISTING

The present application contains a sequence listing that is submittedvia EFS-Web concurrent with the filing of this application, containingthe file name “37759_0206P1_SL.txt” which is 24,576 bytes in size,created on Feb. 6, 2020, and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present disclosure is directed to growth hormone-releasing hormone(GHRH) antagonists and the use of such antagonists for, e.g., inhibitingtumor growth, treating cancer, and/or treating pulmonary fibrosis.

BACKGROUND

Growth hormone-releasing hormone (GHRH) is a peptide belonging to thesecretin/glucagon family of neuroendocrine and gastrointestinalhormones. Human GHRH (hGHRH) peptide comprises 44 amino acid residues.While the best-known site of production of GH-RH is the hypothalamus,various peripheral organs also synthesize it. hGHRH is also produced,sometimes in large quantities, by human malignant tissues (cancers) ofdiverse origin. GHRH exerts various physiological and pathophysiologicalfunctions. There is increasing evidence for the role of GHRH as anautocrine/paracrine growth factor in various cancers. Splice variant(SV) receptors for GHRH, different from those expressed in thepituitary, have been described in a wide range of human cancers and insome normal peripheral organs.

Pathophysiological GH secretion and IGF-1 activation have growthpromoting effects in the lung, and the pituitary type GHRH receptor ispresent in both normal and IPF lung tissue. Idiopathic pulmonaryfibrosis (IPF) is the paradigm of fibrosing interstitial lung diseases.It occurs more commonly in aging males, and often has a limited survivaltime of 3-5 years (median 3.8 years) after diagnosis (Lederer D,Martinez F (2018). N Engl J Med 378:1811-1823). Although the disease isof unknown etiology, it is clearly related to specific geneticabnormalities (e.g., MUC5B, SFTPC and others) and environmental factors(e.g., dust and smoking) (Schwartz D (2016). Trans Am Clin ClimatolAssoc 127:34-45). In response to injury, fibroblasts proliferate andmigrate into the lung. They synthesize extracellular matrix, providing aplatform for further cellular growth (Herrera J, Henke C, Bitterman P(2018). J Clin Invest 128:45-53). Myofibroblasts secrete cytokines, suchas TGF-ß, with autocrine and paracrine effects that drive fibrosis inthe lung (Wei Y, Kim T, Peng D, Duan D, Gibbons D, Yamauchi M, JacksonJ, Le Saux C, Calhoun C, Peters J, Derynck R, Backes B, Chapman H(2017). J Clin Invest 127:3675-3688).

SUMMARY

Disclosed herein are peptides comprising formula I:X0-Tyr-DArg-Asp-Ala-Ile-X6-Thr-X8-X9-X10-X11-X12-Val-Leu-Abu-Gln-Leu-Ser-Ala-X20-X21-Leu-Leu-Gln-Asp-Ile-Nle-DArg-X29-X30(SEQ ID NO: 1), wherein X0 is 5FPhAC-Ada, p-cePhAC, D-Phe-Ada, orPhAC-Ada; X6 is 5FPhe or Cpa; X8 is Ala or Asn; X9 is Arg or Har; X10 isTyr(Me), Amp or 5FPhe; X11 is Arg or His; X12 is Lys or Orn; X20 is Argor His; X21 is Lys or Orn; X29 is Har, Har-NH₂ or Har-NHCH₃; and X30 ispresent or absent and, when present, is Ada-NH₂, Ada-NHCH orAda-NHCH₂CH₃, or a pharmaceutically acceptable salt thereof.

Disclosed herein are methods of treating pulmonary fibrosis, the methodscomprising: administering to a subject with pulmonary fibrosis atherapeutically effective amount of a growth hormone releasing hormone(GHRH) receptor antagonist.

Disclosed herein are methods of reducing lung inflammation, the methodscomprising: administering to a subject with pulmonary fibrosis atherapeutically effective amount of a growth hormone releasing hormone(GHRH) receptor antagonist.

Disclosed herein are methods of reducing lung scarring, the methodscomprising: administering to a subject with pulmonary fibrosis atherapeutically effective amount of a growth hormone releasing hormone(GHRH) antagonist.

Disclosed herein are methods of ameliorating one or more symptoms ofpulmonary fibrosis, the methods comprising: administering to a subjectwith pulmonary fibrosis a therapeutically effective amount of a growthhormone releasing hormone (GHRH) antagonist.

Disclosed herein are methods of reducing expression of one or more Tcell receptor complex genes, the methods comprising administering to asubject with pulmonary fibrosis a therapeutically effective amount of agrowth hormone releasing hormone (GHRH) receptor antagonist.

Disclosed herein are methods of inhibiting tumor growth, the methodscomprising administering to a subject in need thereof an effectiveamount of a growth hormone releasing hormone (GHRH) receptor antagonist.

Other features and advantages of the present compositions and methodsare illustrated in the description below, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1N are graphs showing the effects of GHRH antagonists. FIG. 1Ais a graph showing the effect of GHRH antagonists AVR-333 (5 μg),AVR-352 (2.5 μg), AVR-352 (5 μs), AVR-353 (2.5 μg), AVR-353 (5 μg) andAVR-353 (10 μs) compared to MIA-602 (5 μg) on tumor growth in an animalmodel of pancreatic cancer. Y-axis, % tumor growth; x-axis, time(weeks). FIG. 1B is a graph showing the effect of GHRH antagonistsAVR-235 (5 μg), AVR-333 (5 μg), AVR-353 (5 μg), AVR-553 (5 μg), AVR-543(5 μg) and AVR-352 (5 μg) compared to MIA-602 (5 μg) on tumor growth inan animal model of lung cancer. Y-axis, % tumor growth; x-axis, time(weeks). FIG. 1C is a graph showing the effect of GHRH antagonistsAVR-353 (2 μs), AVR-353 (5 μg) and AVR-353 (10 μg) compared to MIA-602(5 μg) on tumor growth in an animal model of lung cancer. Y-axis, %tumor growth; x-axis, time (weeks). FIG. 1D is a graph showing theeffect of GHRH antagonists MIA-602 (5 μg), AVR-235 (2 μg), AVR-333 (2μg) and AVR-540 (2 μg) compared to MIA-602 (2 μg) on tumor volume in ananimal model of stomach cancer. Y-axis, % tumor growth; x-axis, time(weeks). FIG. 1E is a graph showing the effect of GHRH antagonistsAVR-543 (2 μg), AVR-543 (5 μg), AVR-553 (2 μg) and AVR-553 (5 μg)compared to MIA-602 (5 μg) on tumor growth in an animal model of stomachcancer. Y-axis, % tumor growth; x-axis, time (weeks). FIG. 1F is a graphshowing the effect of GHRH antagonists AVR-235 (2 μg), AVR-353 (2 μg)and AVR-353 (5 μg) compared to MIA-602 (5 μg) on tumor volume in ananimal model of stomach cancer. Y-axis, % tumor growth; x-axis, time(weeks). FIG. 1G is a graph showing the effect of GHRH antagonistsAVR-353 (2 μs), AVR-353 (5 μg) and AVR-354 (5 μg) compared to MIA-602 (5μg) on tumor volume in an animal model of colon cancer. Y-axis, % tumorgrowth; x-axis, time (weeks). FIG. 1H is a graph showing the effect ofGHRH antagonists AVR-352 (5 μg), and AVR-353 (5 μg) compared to MIA-602(5 μg) on tumor growth in an animal model of lung cancer. Y-axis, %tumor growth; x-axis, time (weeks). FIG. 1I is a graph showing theeffect of GHRH antagonists AVR-352 (2.5 μg), AVR-352 (5 μg), AVR-353(2.5 μg), and AVR-353 (5 μg) compared to MIA-602 (5 μg) on tumor growthin an animal model of pancreatic cancer. FIG. 1J is a graph showing theeffect of GHRH antagonists AVR-352 (5 μg), AVR-353 (5 μg) and AVR-354 (5μg) compared to MIA-602 (5 μg) on tumor growth in an animal model ofbreast cancer. FIG. 1K is a graph showing the effect of GHRH antagonistsAVR-352 (5 μg), and AVR-354 (5 μg) compared to MIA-602 (5 μg) on tumorgrowth in an animal model of breast cancer. FIG. 1L is a graph showingthe effect of GHRH antagonists AVR-352 (2.5 μg), AVR-352 (5 μg), AVR-353(2.5 μg), and AVR-353 (5 μg) compared to MIA-602 (5 μg) on tumor growthin an animal model of ovarian cancer. FIG. 1M is a graph showing theeffect of GHRH antagonists AVR-352 (2.5 μg), AVR-352 (5 μg), AVR-353(2.5 μg), and AVR-353 (5 μg) compared to MIA-602 (5 μg) on tumor growthin an animal model of ovarian cancer. FIG. 1N is a graph showing theeffect of GHRH antagonists AVR-352 (5 μg), AVR-353 (5 μg) and AVR-354 (5μg) compared to MIA-602 (5 μg) on tumor growth in an animal model ofprostate cancer. All statistical analyses were performed from comparingGHRH antagonists to non-treated tumor (control). P value of less than0.05 was considered as statistically significant. (Note: *p<0.05,**p<0.01. ***p<0.001.)

FIGS. 2A-F are graphs showing the inhibitory potency of various GHRHantagonists compared to MIA-602. FIG. 2A shows the inhibitory potency ofvarious GHRH antagonists compared to MIA-602 on lung cancer cells(HCC827). FIG. 2B shows the inhibitory potency of various GHRHantagonists compared to MIA-602 on pancreatic cancer cells (CFPAC-1).FIG. 2C shows the inhibitory potency of various GHRH antagonistscompared to MIA-602 on stomach cancer cells (N87). FIG. 2D shows theinhibitory potency of various GHRH antagonists compared to MIA-602 oncolon cancer cells (HT29). FIG. 2E shows the inhibitory potency ofvarious GHRH antagonists compared to MIA-602 on breast cancer cells(MX-1). FIG. 2F shows the inhibitory potency of various GHRH antagonistscompared to MIA-602 on breast cancer cells (HCC1806).

FIG. 3 shows inhibitory effects of GHRH antagonists MIA-602 andAVR-antagonists on the release of GH from rat pituitary cells invitro. * p<0.05, ** p<0.01.

FIG. 4 shows lung hydroxyproline content. Evaluation of lunghydroxyproline (HP) content to estimate changes in collagen due tobleomycin and the effect of MIA-602. Data shown are mean hydroxyprolinecontents of right lungs SEM at 14- and 28-day time points. NormalC57Bl/6J mice (n=6) had about 20 μg HP in the right lung. HP contentincreased significantly (*, P=0.0060 compared to Naive) after 28 days inmice treated with bleomycin and vehicle. No significant increase in HPcontent occurred in lungs of mice treated with bleomycin and the GHRH-Rantagonist MIA-602. M14, MIA-602 group at 14 days (n=5); V14, vehiclegroup at 14 days (n=4); M28, MIA-602 group at 28 days (n=7); V28,vehicle group at 28 days (n=8).

FIG. 5 shows lung histopathology. Mouse lungs were inflated withbuffered formalin to 25 cm H₂O pressure and fixed. Five μm sections werestained with Masson's trichrome stain and assessed semi-quantitativelyfor inflammation and fibrosis as described in the text. Fourteen daysafter bleomycin was started, cellular inflammation and early fibrosiswas detected in lungs of mice treated with bleomycin and vehicle. Lessinflammation appeared to be present in lungs of mice that receivedbleomycin and MIA-602 (middle panels). Twenty-eight days after bleomycinwas started, increased fibrosis was evident in lungs of mice treatedwith bleomycin and vehicle. Less fibrosis appeared to be present inlungs of mice that received bleomycin and MIA-602, the growth hormonereceptor antagonist.

FIGS. 6A-B show that GRHR-R antagonist increases lung fibroblast basaland maximal oxygen consumption. FIG. 6A shows a representativemitochondrial stress assay of mouse lung fibroblasts (data shown aremeans+SD of 6 wells in each condition) exposed to vehicle (light grey),1 μM (medium grey) or 5 μM (black) MIA-602 for 24 hours beforemeasurements of oxygen consumption with oligomycin, FCCP, antimycin Aand rotenone. Five μM MIA-602 increased basal oxygen consumption (*,P=0.0403 compared to vehicle) and maximal, uncoupled respiration (**,P<0.0001) of normal mouse lung fibroblasts. FIG. 6B shows that 5 μMMIA-602 increased both basal respiration (*, P=0.0125) and sparerespiratory capacity (**, P<0.0001).

FIGS. 7A-B show transcriptomic analysis of mouse lung tissue RNA aftertreatment of mice in vivo with bleomycin and MIA-602 or vehicle. FIG. 7Ashows heat map analysis showing differential gene expression in lungsfrom mice treated with bleomycin for 28 days that also received MIA-602(+) or vehicle (−) for the first 21 days. The left two columns show geneexpression in bleomycin- and MIA-602-treated mice, whereas the right twocolumns show gene expression in bleomycin- and vehicle-treated lungs.FIG. 7B shows pathway analysis showing differentially expressed genes(downregulated, top panel; upregulated, bottom panel) in lungs frombleomycin-treated mice also treated with MIA-602 compared to those alsotreated with vehicle.

DETAILED DESCRIPTION

Many modifications and other embodiments of the present disclosure setforth herein will come to mind to one skilled in the art to which thisdisclosure pertains having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the present disclosure is not to be limited to thespecific embodiments disclosed and that modifications and otherembodiments are intended to be included within the scope of the appendedclaims. Although specific terms are employed herein, they are used in ageneric and descriptive sense only and not for purposes of limitation.

Before the present compositions and methods are disclosed and described,it is to be understood that they are not limited to specific syntheticmethods unless otherwise specified, or to particular reagents unlessotherwise specified, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentdisclosure, example methods and materials are now described.

Moreover, it is to be understood that unless otherwise expressly stated,it is in no way intended that any method set forth herein be construedas requiring that its steps be performed in a specific order.Accordingly, where a method claim does not actually recite an order tobe followed by its steps or it is not otherwise specifically stated inthe claims or descriptions that the steps are to be limited to aspecific order, it is in no way intended that an order be inferred, inany respect. This holds for any possible non-express basis forinterpretation, including matters of logic with respect to arrangementof steps or operational flow, plain meaning derived from grammaticalorganization or punctuation, and the number or type of aspects describedin the specification.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present disclosure is not entitled to antedate such publicationby virtue of prior disclosures. Further, the dates of publicationprovided herein can be different from the actual publication dates,which can require independent confirmation.

Definitions

As used in the specification and in the claims, the term “comprising”can include the aspects “consisting of” and “consisting essentially of”“Comprising” can also mean “including but not limited to.”

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” can include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a compound”includes mixtures of compounds; reference to “a pharmaceutical carrier”includes mixtures of two or more such carriers, and the like.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance may or may not occur andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “sample” is meant a tissue or organ from asubject; a cell (either within a subject, taken directly from a subject,or a cell maintained in culture or from a cultured cell line); a celllysate (or lysate fraction) or cell extract; or a solution containingone or more molecules derived from a cell or cellular material (e.g. apolypeptide or nucleic acid), which is assayed as described herein. Asample may also be any body fluid or excretion (for example, but notlimited to, blood, urine, stool, saliva, tears, bile) that containscells or cell components.

As used herein, the term “subject” refers to the target ofadministration, e.g., a human. Thus, the subject of the disclosedmethods can be a vertebrate, such as a mammal, a fish, a bird, areptile, or an amphibian. The term “subject” also includes domesticatedanimals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs,sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat,guinea pig, fruit fly, etc.). In some aspects, a subject can be amammal. In some aspects, a subject can a human. The term does not denotea particular age or sex. Thus, adult, child, adolescent and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

As used herein, the term “patient” refers to a subject afflicted with adisease or disorder. The term “patient” includes human and veterinarysubjects. In some aspects of the disclosed methods, the “patient” hasbeen diagnosed with a need for treatment for pulmonary fibrosis, suchas, for example, prior to the administering step. In some aspects of thedisclosed methods, the “patient” has been diagnosed with a need fortreatment for cancer, such as, for example, prior to the administeringstep.

Ranges can be expressed herein as from “about” or “approximately” oneparticular value, and/or to “about” or “approximately” anotherparticular value. When such a range is expressed, a further aspectincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” or “approximately,” it will be understood thatthe particular value forms a further aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein and that each value is also herein disclosed as “about”that particular value in addition to the value itself. For example, ifthe value “10” is disclosed, then “about 10” is also disclosed. It isalso understood that each unit between two particular units is alsodisclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and14 are also disclosed.

As used herein, the term “treating” refers to partially or completelyalleviating, ameliorating, relieving, delaying onset of, inhibiting orslowing progression of, reducing severity of, and/or reducing incidenceof one or more symptoms or features of a particular disease, disorder,and/or condition. Treatment can be administered to a subject who doesnot exhibit signs of a disease, disorder, and/or condition and/or to asubject who exhibits only early signs of a disease, disorder, and/orcondition for the purpose of decreasing the risk of developing pathologyassociated with the disease, disorder, and/or condition. Treatment canalso be administered to a subject to ameliorate one more signs ofsymptoms of a disease, disorder, and/or condition. For example, thedisease, disorder, and/or condition can be pulmonary fibrosis or acancer.

The term “fragment” can refer to a portion (e.g., at least 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, etc. amino acids) of a peptide that is substantiallyidentical to a reference peptide and retains the biological activity ofthe reference. In some aspects, the fragment or portion retains at least50%, 75%, 80%, 85%, 90%, 95% or 99% of the biological activity of thereference peptide described herein. Further, a fragment of a referencedpeptide can be a continuous or contiguous portion of the referencedpolypeptide (e.g., a fragment of a peptide that is ten amino acids longcan be any 2-9 contiguous residues within that peptide).

A “variant” can mean a difference in some way from the referencesequence other than just a simple deletion of an N- and/or C-terminalamino acid residue or residues. For example, disclosed are variants ofthe growth hormone releasing hormone peptides described herein. Wherethe variant includes a substitution of an amino acid residue, thesubstitution can be considered conservative or non-conservative.Conservative substitutions are those within the following groups: Ser,Thr, and Cys; Leu, ILe, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, andTrp; and Gln, Asn, Glu, Asp, and His. Variants can include at least onesubstitution and/or at least one addition, there may also be at leastone deletion. Variants can also include one or more non-naturallyoccurring residues. For example, they may include selenocysteine (e.g.,seleno-L-cysteine) at any position, including in the place of cysteine.Many other “unnatural” amino acid substitutes are known in the art andare available from commercial sources. Examples of non-naturallyoccurring amino acids include D-amino acids, amino acid residues havingan acetylaminomethyl group attached to a sulfur atom of a cysteine, apegylated amino acid, and omega amino acids of the formula NH₂(CH₂)nCOOHwherein n is 2-6 neutral, nonpolar amino acids, such as sarcosine,t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine.Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline andmethionine sulfoxide are neutral nonpolar, cysteic acid is acidic, andomithine is basic. Proline may be substituted with hydroxyproline andretain the conformation conferring properties of proline. In someaspects, the variants can comprise a sequence having at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity to any of thesequences described herein. In some aspects, the variants retain atleast 50%, 75%, 80%, 85%, 90%, 95% or 99% of the biological activity ofthe reference peptide described herein.

As used herein, the term “amelioration” refers to a lessening of atleast one indicator, sign, or symptom of an associated disease,disorder, or condition. The severity of indicators may be determined bysubjective or objective measures, which are known to those skilled inthe art.

As used herein the terms “amino acid” and “amino acid identity” refersto one of the 20 naturally occurring amino acids or any non-naturalanalogues that may be in any of the antibodies, variants, or fragmentsdisclosed. Thus “amino acid” as used herein means both naturallyoccurring and synthetic amino acids. For example, homophenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chain may be in either the (R) orthe (S) configuration. In some aspects, the amino acids are in the (S)or L-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradation.

“Inhibit,” “inhibiting” and “inhibition” mean to diminish or decrease anactivity, response, condition, disease, or other biological parameter.This can include, but is not limited to, the complete ablation of theactivity, response, condition, or disease. This may also include, forexample, a 10% inhibition or reduction in the activity, response,condition, or disease as compared to the native or control level. Thus,in an aspect, the inhibition or reduction can be a 10, 20, 30, 40, 50,60, 70, 80, 90, 100%, or any amount of reduction in between as comparedto native or control levels. In an aspect, the inhibition or reductionis 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% ascompared to native or control levels. In an aspect, the inhibition orreduction is 0-25, 25-50, 50-75, or 75-100% as compared to native orcontrol levels.

As used herein, the term “prevent” or “preventing” refers to preventingin whole or in part, or ameliorating or controlling.

Abbreviations

Amino Acid Abbreviations 12-aminododecanoic acid Ada aminobutyric acidAbu alanine Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acidAsp (D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (K) glycineGly (G) histidine His (H) homoarginine Har isolelucine Ile (I) leucineLeu (L) lysine Lys (K) methionine Met (M) norleucine Nle ornithine Ornphenylalanine Phe (F) proline Pro (P) serine Ser (S) threonine Thr (T)tyrosine Tyr (Y) tryptophan Trp (W) valine Val (V_(—) Phenylacetic acidPhAC Pentafluorophenylacetic 5FPhAC acid Pentafluorophenylalanine 5FPhe8-aminooctanotic acid Aoc

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

The present disclosure is based in part on the discovery that certaingrowth hormone-releasing hormone (GHRH) antagonists have an inhibitoryeffect on several human cancers, including human gastric, pancreatic,colorectal and lung cancers.

Growth hormone-releasing hormone (GHRH) is secreted primarily from thehypothalamus, but various other tissues can produce it locally (KiarisH, Chatzistamou I, Papavassiliou A, Schally A (2011). Trends EndocrinolMetab 22:311-317). GHRH stimulates the secretion and release of growthhormone (GH) by the pituitary and in turn regulates the secretion of GHand insulin-like growth factor 1 (IGF-1) through the pituitaryGH/hepatic IGF-1 axis (Hung C, Rohani M, Lee S, Chen P, Schnapp L(2013). Respir Res 14:102). Expression of pituitary type GHRH-receptor(pGHRH-R) has been found in normal human and IPF lung tissue by westernblotting, suggesting that GHRH or GH could participate in lungdevelopment, growth and repair (Jackson R, Ai L, Zhang C, Zhang X,Delcroix G, Lazerson A, Mirsaeidi M, Schally A (2018). EuropeanRespiratory Journal 52 (suppl 62): OA5349).

GHRH belongs to a peptide family that includes glucagon, secretin,vasoactive intestinal peptide (VIP), and pituitary adenylatecyclase-activating peptide (PACAP) (Kiaris H, Chatzistamou 1,Papavassiliou A, Schally A (2011). Trends Endocrinol Metab 22:311-317).GHRH-R antagonists exert growth-inhibitory effects in cancers in vitroand in vivo (Perez R, Schally A, Vidaurre I, Ricon R, Block N, Rick F(2012). Oncotarget (3):988-997; Schally A, Varga J, Engel J (2007).Nature Clin Pract Endo Metab 4:33-43; and Zarandi M, Cai R, Kovacs M,Popovics P, Szalontay L, Cui T, Sha W, Jaszberenyi M, Varga J, Zhang X,Block N, Rick F, Halmos G, Schally A (2017). Peptides 89:60-70), inaddition to having anti-inflammatory and anti-oxidative effects(Barbutis N, Schally A (2008). PNAS 105:20470-20475).

Human fibroblasts express GHRH receptors, which stimulate proliferationof fibroblasts through GH/IGF-1-mediated signaling. When skin wounds inmice are exposed to GHRH agonist, fibroblasts increase and repair ofepithelium is accelerated (Cui T, Jimenez J, Block N, Badiavas E,Rodriguez-Menocal L, Vila Granda A, Cai R, Sha W, Zarandi M, Perez R,Schally A (2016). Oncotarget 7: 52661-52672). GHRH stimulates theexpression of α-smooth muscle actin (αSMA), which confers contractileactivity in myofibroblasts (Zhang X, Xing R, Chen L, Liu C, Miao Z(2016). Molecular Medicine Reports 14(6):5699-570). In addition to itseffects on GH and IGF-1, GHRH-R antagonist MIA-602 inhibits signalingpathways, including PAK1-STAT3/NF-κB in gastric cancer cells, suggestingit could modulate inflammatory and fibrotic processes (Gan J, Ke X,Jiang J, Dong H. Yao Z, Lin Y, Lin W, Wu X, Yan S, Zhuang Y, Chu W, CaiR, Zhang X, Cheung H, Block N, Pang C, Schally A, Zhang H (2016). ProcNatl Acad Sci USA 113:14745-14750).

Described herein is the use of an established bleomycin model of lunginflammation and fibrosis in C57/B16 mice and synthetic GHRH receptorantagonist MIA-602 to test whether inhibition of GHRH receptors wouldlimit inflammation and/or fibrosis.

Disclosed herein are methods of synthesizing highly potent antagonistsof growth hormone-releasing hormone (GHRH) AVR class, and methods forusing them to inhibit tumor growth, lung inflammation and/or fibrosis.

Compositions

Disclosed herein are growth hormone releasing hormone (GHRH) receptorantagonists (GHRH-R antagonists). In some aspects, the growth hormonereleasing hormone (GHRH) receptor antagonists are peptides. In someaspects, the growth hormone releasing hormone (GHRH) receptorantagonists are growth hormone releasing peptides. For example, MIA-602is a GHRH-R antagonist having the following amino acid sequence,Tyr-Ala-Asp-Ala-Ile⁵-Phe-Thr-Asn-Ser-Tyr¹⁰-Arg-Lys-Val-Leu-Gly¹⁵-Gln-Leu-Ser-Ala-Arg²⁰-Lys-Leu-Leu-Gln-Asp²⁵-Tle-Met-Ser-Arg²⁹-NH₂(SEQ ID NO: 21). MTA-602 can also be referred to as hGH-RH(1-29)NH₂.hGH-RH(1-29)NH₂ I is considered a standard GHRH-R antagonist[Ac-Tyr¹,D-Arg²]. hGH-RH(1-29)NH₂ is fragment of the native GH-RH.Synthetic analogs of GHRH based on the structure of hGH-RH(1-29)NH₂ (SEQID NO: 21) can be used in the methods disclosed herein. Examples of GHRHanalogs are disclosed in U.S. Pat. No. 9,260,504 and are incorporatedherein by reference. Also disclosed are variants of MIA-602 andfragments thereof.

Disclosed herein are growth hormone releasing hormone (GHRH) receptorantagonists that can be used in methods of treating pulmonary fibrosis;reducing lung inflammation; reducing lung scarring; ameliorating one ormore symptoms of pulmonary fibrosis; and reducing expression of one ormore T cell receptor complex genes.

In some aspects, the GHRH receptor antagonist can be a growth hormonereleasing hormone peptide. In some aspects, the growth hormone releasinghormone peptide comprises or consists of:

(a) (AVR-235, SEQ ID NO: 3)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-NHCH₃; (b) (AVR-333, SEQ ID NO: 4)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂; (c)(AVR-352, SEQ ID NO: 5) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada- NH₂; (d)(AVR-353, SEQ ID NO: 6) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂; or (e)(AVR-354, SEQ ID NO: 7) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃.Also disclosed are variants of these growth hormone releasing hormonepeptides.

In some aspects, the growth hormone releasing hormone peptide comprisesor consists of:

(a)  (AVR-104, SEQ ID NO: 8)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂; (b)  (AVR-107, SEQ ID NO: 9)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂; (c)  (AVR-116, SEQ ID NO: 10)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂; (d)  (AVR-120, SEQ ID NO: 11)D-Phe-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂; (e)  (AVR-201, SEQ ID NO: 12)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Amp-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃; (f)  (AVR-234, SEQ ID NO: 13)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃; (g) (AVR-321, SEQ ID NO: 14) PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Aoc-NHCH₃; (h) (AVR-322, SEQ ID NO: 15)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Aoc-NHCH₃; (i) (AVR-542, SEQ ID NO: 16)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃; (j) (AVR-543, SEQ ID NO: 17)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NHCH₃; (k) (AVR-552, SEQ ID NO: 18)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂; (l) (AVR-553, SEQ ID NO: 19) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada- NH₂; or (m) (AVR-620, SEQ ID NO: 20)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂.Also disclosed are variants of these growth hormone releasing hormonepeptides.

In some aspects, the growth hormone releasing hormone peptide or GHRHreceptor antagonist is not MIA-602, SEQ ID NO: 21. In some aspects, thegrowth hormone releasing hormone peptide or GHRH receptor antagonistdoes not comprise the sequence of MIA-602, SEQ ID NO: 21

Described herein is a GHRH antagonist comprising the amino acid sequence(formula I):X0-Tyr-DArg-Asp-Ala-Ile-X6-Thr-X8-X9-X10-X11-X12-Val-Leu-Abu-Gln-Leu-Ser-Ala-X20-X21-Leu-Leu-Gln-Asp-Ile-Nle-DArg-X29-X30(SEQ ID NO: 1). In some aspects, X0 can be 5FPhAC-Ada, D-Phe-Ada,P-ClPhAC, or PhAC-Ada; X6 can be 5FPhe or Cpa; X8 can be Ala or Asn; X9can be Arg or Har; X10 can be Tyr(Me), 5FPhe or Amp; X11 can be Arg orHis; X12 can be Lys or Orn; X20 can be Arg or His; X21 can be Lys orOrn; X29 can be Har, Har-NH₂ or Har-NHCH₃; and X30 can be present orabsent and, when present, can be Ada-NH₂, Aoc-NHCH₃ or Ada-NHCH₃; or apharmaceutically acceptable salt thereof.

Described herein is a GHRH antagonist comprising the amino acid sequence(formula II):5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-X6-Thr-X8-Har-X10-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-X29-X30(SEQ ID NO: 2). In some aspects, X6 can be 5FPhe or Cpa; X8 can be Alaor Asn; X10 can be Tyr(Me) or 5FPhe; X29 can be Har or Har-NHCH₃; andX30 can be present or absent and, when present, can be Ada-NH₂ orAda-NHCH₃; or a pharmaceutically acceptable salt thereof.

Described herein is a GHRH antagonist comprising the amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-NHCH₃(AVR-235, SEQ ID NO: 3). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂(AVR-333, SEQ ID NO: 4). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂(AVR-352, SEQ ID NO: 5). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂(AVR-353, SEQ ID NO: 6). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Har-NHCH₃(AVR-354, SEQ ID NO: 7). In some aspects, the GHRH antagonist comprisesthe amino acid sequencePhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Om-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂(AVR-104, SEQ ID NO: 8). In some aspects, the GHRH antagonist comprisesthe amino acid sequencePhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂(AVR-107, SEQ ID NO: 9). In some aspects, the GHRH antagonist comprisesthe amino acid sequencePhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂(AVR-116, SEQ ID NO: 10). In some aspects, the GHRH antagonist comprisesthe amino acid sequenceD-Phe-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂(AVR-120, SEQ ID NO: 11). In some aspects, the GHRH antagonist comprisesthe amino acid sequencePhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Amp-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃(AVR-201, SEQ ID NO: 12). In some aspects, the GHRH antagonist comprisesthe amino acid sequencePhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃(AVR-234, SEQ ID NO: 13). In some aspects, the GHRH antagonist comprisesthe amino acid sequencePhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Aoc-NHCH₃(AVR-321, SEQ ID NO: 14). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Aoc-NHCH₃(AVR-322, SEQ ID NO: 15). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃(AVR-542, SEQ ID NO: 16). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NHCH₃(AVR-543, SEQ ID NO: 17). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂(AVR-552, SEQ ID NO: 18). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂(AVR-553, SEQ ID NO: 19). In some aspects, the GHRH antagonist comprisesthe amino acid sequence5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂(AVR-620, SEQ ID NO: 20).

As used herein, the term “peptide” refers to a linear molecule formed bybinding amino acid residues to each other via peptide bonds. As usedherein, the term “polypeptide” refers to a polymer of (the same ordifferent) amino acids bound to each other via peptide bonds.

Analogs, fragments and variants of GHRH and any of the growth hormonereleasing hormone peptides described herein can be synthesized usingstandard techniques of peptide chemistry.

In some aspects, the growth hormone releasing hormone peptides describedherein can be further modified to improve stability. In some aspects,any of the amino acid residues of the growth hormone releasing hormonepeptides described herein can be modified to improve stability. In someaspects, growth hormone releasing hormone peptides can have at least oneamino acid residue that has an acetyl group, a fluorenylmethoxy carbonylgroup, a formyl group, a palmitoyl group, a myristyl group, a stearylgroup, or polyethylene glycol. In some aspects, an acetyl protectivegroup can be bound to the growth hormone releasing hormone peptidesdescribed herein.

As used herein, the term “stability” refers to storage stability (e.g.,room-temperature stability) as well as in vivo stability. The foregoingprotective group can protect the peptides described herein from theattack of protein cleavage enzymes in vivo.

As used herein, the term “growth hormone releasing hormone peptide” canalso be used to include functional equivalents of the growth hormonereleasing hormone peptides described herein or variants thereof. As usedherein, the term “functional equivalents” can refer to amino acidsequence variants having an amino acid substitution, addition, ordeletion in some of the amino acid sequence of the growth hormonereleasing hormone peptides while simultaneously having similar orimproved biological activity, compared with the growth hormone releasinghormone peptides as described herein. In some aspects, the amino acidsubstitution can be a conservative substitution. Examples of thenaturally occurring amino acid conservative substitution include, forexample, aliphatic amino acids (Gly, Ala, and Pro), hydrophobic aminoacids (Ile, Leu, and Val), aromatic amino acids (Phe, Tyr, and Trp),acidic amino acids (Asp and Glu), basic amino acids (His, Lys, Arg, Gln,and Asn), and sulfur-containing amino acids (Cys and Met).

Any of the compositions disclosed herein can further comprise apharmaceutically acceptable carrier. In some aspects, thepharmaceutically acceptable carrier for the growth hormone releasinghormone peptides can be buffered saline. In some aspects, thepharmaceutically acceptable carrier for the small molecule can be wateror DMSO. In some aspects, the pharmaceutically acceptable carrier cancomprise a lipid-based or polymer-based colloid. In some aspects, thecolloid can be a liposome, a hydrogel, a microparticle, a nanoparticle,or a block copolymer micelle. In some aspects, the compositionsdescribed herein can be formulated for intravenous, subcutaneous,intrathecal, intratracheal, intramuscular, oral or intraperitonealadministration.

Methods of Treatment

Disclosed herein are methods of inhibiting tumor growth. In someaspects, the methods can comprise administering to a subject in needthereof an effective amount of any one of the GHRH antagonists describedherein. In some aspects, the tumor can be prostate cancer, breastcancer, lung cancer, colorectal cancer, melanoma, bladder cancer,brain/CNS cancer, cervical cancer, esophageal cancer, stomach cancer,colon cancer, head/neck cancer, kidney cancer, liver cancer, lymphoma,ovarian cancer, pancreatic cancer, thyroid cancer, glioblastoma,leukemia or sarcoma. In some aspects, the tumor can be a primary tumoror a metastatic tumor.

In some aspects, any of the GHRH antagonists described herein caninhibit growth of a tumor associated with any cancer type. Examples ofcancers include, but are not limited to, adrenocortical carcinoma,AIDS-related cancers, AIDS-related lymphoma, anal cancer, anorectalcancer, cancer of the anal canal, appendix cancer, childhood cerebellarastrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skincancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer,intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer,bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma,brain cancer, brain tumor, brain stem glioma, glioblastoma, cerebellarastrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,medulloblastoma, supratentorial primitive neuroectodeimal tumors, visualpathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer,laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia,chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cellleukemia, lip and oral cavity cancer, liver cancer, lung cancer,non-small cell lung cancer, small cell lung cancer, AIDS-relatedlymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma,Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular(eye) melanoma, merkel cell carcinoma, mesothelioma malignant,mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer ofthe tongue, multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterinecancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer(melanoma), merkel cell skin carcinoma, small intestine cancer, softtissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, testicular cancer,throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter and otherurinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, and Wilm's Tumor. In some aspects, thetumor can be associated with a cancer selected from the group consistingof breast cancer, melanoma, prostate cancer, pancreatic cancer, head andneck cancer, lung cancer, non small-cell lung carcinoma, renal cancer,colorectal cancer, bladder cancer, stomach cancer, ovarian cancer,sarcoma, esophageal cancer, cervical cancer and gastric cancer. In someaspects, the cancer can be pancreatic cancer.

The disclosure also contemplates use of any of the GHRH antagonistsdescribed herein to inhibit tumor growth or treat cancer, as well as useof any of the GHRH antagonist described herein in the preparation of amedicament for inhibiting tumor growth or treating cancer.

Also disclosed herein are methods of treating cancer. In some aspects,the methods can comprise administering to a subject in need thereof aneffective amount of any one of the GHRH antagonists described herein. Insome aspects, the cancer can be prostate cancer, breast cancer, lungcancer, colorectal cancer, melanoma, bladder cancer, brain/CNS cancer,cervical cancer, esophageal cancer, stomach cancer, colon cancer,head/neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer,pancreatic cancer, thyroid cancer, glioblastoma, leukemia or sarcoma.

Further disclosed herein are methods of treating pulmonary fibrosiscomprising: administering to a subject with pulmonary fibrosis atherapeutically effective amount of a growth hormone releasing hormone(GHRH) receptor antagonist. Also disclosed herein are methods ofreducing lung inflammation comprising: administering to a subject withpulmonary fibrosis a therapeutically effective amount of a growthhormone releasing hormone (GHRH) receptor antagonist. Lung inflammationcan be characterized by infiltration of lung tissue with one or moreinflammatory cells resulting in impaired oxygen uptake and one or moresymptoms such as breathlessness and cough. Further, lung injury causedby inflammation can cause disordered healing and scar formation, whichare also characteristic of pulmonary fibrosis. Further disclosed hereinare methods of reducing lung scarring comprising: administering to asubject with pulmonary fibrosis a therapeutically effective amount of agrowth hormone releasing hormone (GHRH) receptor antagonist. Alsodisclosed herein are of methods of ameliorating one or more symptoms ofpulmonary fibrosis comprising: administering to a subject with pulmonaryfibrosis a therapeutically effective amount of a growth hormonereleasing hormone (GHRH) receptor antagonist. In some aspects, the oneor more symptoms of pulmonary fibrosis can be breathlessness, cough,decreased exercise tolerance, hypoxemia or a combination thereof.

Disclosed herein are methods of reducing expression of one or more Tcell receptor complex genes comprising: administering to a subject withpulmonary fibrosis a therapeutically effective amount of a growthhormone releasing hormone (GHRH) receptor antagonist. In some aspects,the one or more T cell receptor complex genes can be CD3E, CD3G, CD4, orCD8A.

In some aspects, the method can comprise: administering to a subjectwith pulmonary fibrosis a therapeutically effective amount of a growthhormone releasing hormone (GHRH) receptor antagonist. In some aspects,the GHRH receptor antagonist can be a growth hormone releasing hormonepeptide. In some aspects, the growth hormone releasing hormone peptideor GHRH receptor antagonist is not MIA-602 (SEQ ID NO: 21).

In some aspects, the growth hormone releasing hormone peptide used inthe methods disclosed herein comprises the amino acid sequence:

(a) (AVR-235, SEQ ID NO: 3)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-NHCH₃, (b) (AVR-333, SEQ ID NO: 4)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂; (c)(AVR-352, SEQ ID NO: 5) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂; (d)(AVR-353, SEQ ID NO: 6) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂; or (e)(AVR-354, SEQ ID NO: 7) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃or variants or fragments thereof.

In some aspects, the growth hormone releasing hormone peptide comprisesthe amino acid sequence:

(a)  (AVR-104, SEQ ID NO: 8)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂; (b)  (AVR-107, SEQ ID NO: 9)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂; (c)  (AVR-116, SEQ ID NO: 10)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂; (d)  (AVR-120, SEQ ID NO: 11)D-Phe-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH₂; (e)  (AVR-201, SEQ ID NO: 12)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Amp-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃; (f)  (AVR-234, SEQ ID NO: 13)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃; (g) (AVR-321, SEQ ID NO: 14) PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Aoc-NHCH₃; (h) (AVR-322, SEQ ID NO: 15)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Aoc-NHCH₃; (i) (AVR-542, SEQ ID NO: 16)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Len-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH₃; (j) (AVR-543, SEQ ID NO: 17)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Len-Gln-Asp-Ile-Nle-DArg-Har-Ada-NHCH₃; (k) (AVR-552, SEQ ID NO: 18)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂; (l) (AVR-553, SEQ ID NO: 19) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada- NH₂; or (m) (AVR-620, SEQ ID NO: 20)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada-NH₂or variants or fragments thereof.

In some aspects, the methods can further include the step of identifyinga subject (e.g., a human patient) who has or is at risk for havingpulmonary fibrosis and then providing to the subject a compositioncomprising a therapeutically effective amount of a growth hormonereleasing hormone (GHRH) receptor antagonist. In some aspects, the GHRHreceptor antagonist is a growth hormone releasing hormone peptide. Insome aspects, the subject can be identified using standard clinicaltests known to those skilled in the art. Examples of tests fordiagnosing pulmonary fibrosis include but are not limited to highresolution computed tomographic (HRCT) scans of the chest,video-assisted thoracoscopic (VATS) lung biopsies, pulmonary functiontests (PFT) and multidisciplinary consultations.

The therapeutically effective amount can be the amount of thecomposition administered to a subject that leads to a full resolution ofthe symptoms of the condition or disease, a reduction in the severity ofthe symptoms of the condition or disease, or a slowing of theprogression of symptoms of the condition or disease. The methodsdescribed herein can also include a monitoring step to optimize dosing.The compositions described herein can be administered as a preventivetreatment or to delay or slow the progression of pulmonary fibrosis.

The dosage to be administered depends on many factors including, forexample, the route of administration, the formulation, the severity ofthe patient's condition/disease, previous treatments, the patient'ssize, weight, surface area, age, and gender, other drugs beingadministered, and the overall general health of the patient includingthe presence or absence of other diseases, disorders or illnesses.Dosage levels can be adjusted using standard empirical methods foroptimization known by one skilled in the art. Administrations of thecompositions described herein can be single or multiple (e.g., 2- or 3-,4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Further,encapsulation of the compositions in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) can improve theefficiency of delivery.

The therapeutically effective amount of the compositions describedherein can include a single treatment or a series of treatments (i.e.,multiple treatments or administered multiple times). Treatment durationusing any of compositions disclosed herein can be any length of time,such as, for example, one day to as long as the life span of the subject(e.g., many years). For instance, the composition can be administereddaily, weekly, monthly, yearly for a period of 5 years, ten years, orlonger. The frequency of treatment can vary. For example, thecompositions described herein can be administered once (or twice, threetimes, etc.) daily, weekly, monthly, or yearly for a period of 5 years,ten years, or longer.

Combination Therapy

In some aspects, the compositions disclosed herein can also beco-administered with another therapeutic agent. Combination therapy (or“co-therapy”) can include the GHRH antagonist and another agent as partof a specific treatment regimen intended to provide the beneficialeffect from the combined action of these therapeutic agents. Additionaltherapeutic agents or therapies contemplated for use with the GHRHantagonist described herein include, but are not limited to, androgendeprivation therapy, a chemotherapeutic agent, a radiotherapeutic agent,an immunotherapeutic agent, an inhibitor of cellular proliferation, aregulator of programmed cell death, surgery and other agents.

Androgen deprivation therapy. In some aspects, androgen deprivationtherapy can be administered to the subject in combination with the GHRHantagonist. Androgen deprivation therapy comprises the administration ofan inhibitor of androgen synthesis to the subject, administration of anandrogen receptor antagonist to the subject, administration of agonadotropin-releasing hormone (GnRH) agonist, administration of a GnRHantagonist or a combination thereof.

In some aspects, the methods described herein further compriseadministering an androgen receptor antagonist to the subject. Examplesof androgen receptor antagonists include, but are not limited to,Enzalutamide, Bicalutamide, Ostarine, Flutamide, Cyproterone acetate,Gugguisterone, Nilutamide, PF998245, (R)-Bicalutamide, and1,1-Dichloro-2,2-bis(4-chlorophenyl)ethene, ARN-509 and MDV-3100.

In some aspects, the methods described herein can further compriseadministering an inhibitor of androgen synthesis to the subject. In someaspects, the inhibitor of androgen synthesis can be Abiraterone acetate.

In some aspects, the methods described herein further compriseadministering a GnRH agonist to the subject. Examples of GnRH agonistsinclude, but are not limited to, leuprolide, buserelin, histrelin,goserelin and deslorelin.

In some aspects, the methods described herein further compriseadministering a GnRH antagonist to the subject. Examples GnRHantagonists include, but are not limited to, cetrorelix, ganirelix,abarelix and degarelix.

Chemotherapeutic Agents. In some aspects, chemotherapy may beadministered, optionally in regular cycles. Standard of carechemotherapeutic regimens for patients with prostate cancer include, butare not limited to docetazel, cabazitaxel, mitoxantrone, estramustine,doxorubicin, etoposide, vinblastine, paclitaxel, carboplatin andvinorelbine. In some aspects, docetaxel in combination with predisonecan be administered in combination with any of the GHRH antagonistdescribed herein.

Chemotherapeutic agents contemplated for use with the methods describedherein, include, but are not limited, to erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), bevacizumab (AVASTTN®, Genentech), trastuzumab(HERCEPTIN®, Genentech), pertuzumab (OMNITARG®, rhuMab 2C4, Genentech),temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carbox-amide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanam-ine,NOLVADEX®, ISTUBAL®, VALODEX®), doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, rapamycin, and lapatinib (TYKERB®, Glaxo SmithKline), oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (MEK inhibitor, Exelixis, WO2007/044515), ARRY-886 (MEK inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), ABT-869(multi-targeted inhibitor of VEGF and PDGF family receptor tyrosinekinases, Abbott Laboratories and Genentech), ABT-263 (Bcl-2/Bcl-xLinhibitor, Abbott Laboratories and Genentech), PTK787/ZK 222584(Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinicacid), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib(NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca),irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson& Johnson); capecitabine (XELODA®, Roche), ABRAXANE™ (Cremophor-free),albumin-engineered nanoparticle formulations of paclitaxel (AmericanPharmaceutical Partners, Schaumberg, Ill.), vandetanib (rINN. ZD6474,ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen),temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline),canfosfamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide(CYTOXAN®, NEOSAR®), alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone), a camptothecin (including thesynthetic analog topotecan), bryostatin, callystatin, CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8),dolastatin, duocarmycin (including the synthetic analogs, KW-2189 andCBI-TMI); eleutherobin, pancratistatin, a sarcodictyin; spongistatin,nitrogen mustards such as chlorambucil, chlomaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gammalI, calicheamicin omegaI1, dynemicin,dynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin;olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids andderivatives of any of the above.

Radiation therapy. Radiation and radiotherapeutic agents may also beused in accordance with the methods described herein. Radiationincludes, e.g., 7-rays, X-rays, microwaves and UV-irradiation. Radiationmay be applied directly to an area of interest by directed delivery ofradioisotopes to tumor cells. It is most likely that any of thesefactors can effect a broad range of damage on DNA, on the precursors ofDNA, on the replication and repair of DNA, and/or on the assembly andmaintenance of chromosomes. Dosage ranges for X-rays range from dailydoses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk),to single doses of 2000 to 6000 roentgens. Dosage ranges forradioisotopes vary widely, and depend on the half-life of the isotope,the strength and type of radiation emitted, and the uptake by theneoplastic cells.

Immunotherapeutic agents. Immunotherapeutics may also be employed forthe treatment of cancer. Immunotherapeutics, generally, rely on the useof immune effector cells and molecules to target and destroy cancercells. The immune effector may be, for example, an antibody specific forsome marker on the surface of a tumor cell. The antibody alone may serveas an effector of therapy or it may recruit other cells to effect cellkilling. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells.

Generally, for immunotherapy, the tumor cell must bear some marker thatis amenable to targeting, i.e., is not present on the majority of othercells. Many tumor markers exist and any of these may be suitable fortargeting in the context of the present invention. Examples of markersexpressed in prostate tissues include, but are not limited to,prostate-specific antigen (PSA), prostate-specific membrane antigen(PSMA), prostatic acid phosphatase (PAP), prostate stem cell antigen(PSCA), T cell receptor gamma alternate reading frame protein (TARP),transient receptor potential (trp)-p8 and six-transmembrane epithelialantigen of the prostate 1 (STEAP1).

Regulators of programmed cell death. Apoptosis, or programmed celldeath, is an important process in cancer therapy (Kerr et al., 1972).The Bcl-2 family of proteins and ICE-like proteases have beendemonstrated to be important regulators and effectors of apoptosis inother systems. The Bcl-2 protein, discovered in association withfollicular lymphoma, plays a prominent role in controlling apoptosis andenhancing cell survival in response to diverse apoptotic stimuli(Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et al., 1986;Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarilyconserved Bcl-2 protein now is recognized to be a member of a family ofrelated proteins, which can be categorized as death agonists or deathantagonists.

Members of the Bcl-2 that function to promote cell death such as, Bax,Bak, Bik, Bim, Bid, Bad and Harakiri, are contemplated for use incombination the AVPR antagonist described herein.

Surgery. In some aspects, a surgical procedure may be employed.Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, and microscopicallycontrolled surgery (Mohs' surgery). In some aspects, any of thecompositions and methods described herein can be used in conjunctionwith removal of superficial cancers, precancers, or incidental amountsof normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with any ofthe GHRH antagonists described herein. In some aspects, the treatmentmay be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, orevery 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 months. These treatments may be of varying dosages as well.

Other agents. In some aspects, other agents may be used in combinationwith the methods described herein to improve the therapeutic efficacy oftreatment. These additional agents include immunomodulatory agents,agents that affect the upregulation of cell surface receptors and GAPjunctions, cytostatic and differentiation agents, inhibitors of celladhesion, or agents that increase the sensitivity of thehyperproliferative cells to apoptotic inducers. Immunomodulatory agentsinclude tumor necrosis factor; interferon alpha, beta, and gamma; IL-2and other cytokines; F42K and other cytokine analogs; or MIP-1,MIP-1beta, MCP-1, RANTES, and other chemokines. In some aspects, theupregulation of cell surface receptors or their ligands such as Fas/Fasligand, DR4 or DR5/TRAIL can potentiate the apoptotic inducing abilitiesof the present invention by establishment of an autocrine or paracrineeffect on hyperproliferative cells. Increased intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In some aspects, cytostatic or differentiation agentscan be used in combination with the invention to improve theanti-hyperproliferative efficacy of the treatments. Inhibitors of celladhesion can also be administered to improve the efficacy of treatment.In some aspects, the cell adhesion inhibitor can be a focal adhesionkinase (FAK) inhibitor or lovastatin.

Pulmonary fibrosis or lung cancer. In some aspects, the methodsdisclosed herein can further comprise administering pirfenidone(Esbriet®) or nintedanib (Ofev® and Vargatef®) to the subject. In someaspects, the methods disclosed herein can further comprise administeringan anti-inflammatory therapy to the subject.

Pharmaceutical Compositions

As disclosed herein, are pharmaceutical compositions, comprising thecompositions disclosed herein. In an aspect, the pharmaceuticalcomposition can comprise any of the growth hormone releasing hormonepeptides, fragments of the growth hormone releasing hormone peptides orvariants of the growth hormone releasing hormone peptides disclosedherein. In some aspects, the pharmaceutical compositions can furthercomprise a pharmaceutically acceptable carrier. In some aspects, thepharmaceutical compositions described herein can be sterile and containany of the GHRH antagonists for producing the desired response in a unitof weight or volume suitable for administration to a subject. In someaspects, the pharmaceutical compositions can contain suitable bufferingagents, including, e.g., acetic acid in a salt; citric acid in a salt;boric acid in a salt; and phosphoric acid in a salt.

When administered, the therapeutic composition(s) can be administered inpharmaceutically acceptable preparations. Such preparations mayroutinely contain pharmaceutically acceptable concentrations of salt,buffering agents, preservatives, compatible carriers, supplementaryimmune potentiating agents such as adjuvants and cytokines, andoptionally other therapeutic agents.

As used herein, the term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. The term “physiologicallyacceptable” refers to a non-toxic material that is compatible with abiological system such as a cell, cell culture, tissue, or organism. Thecharacteristics of the carrier will depend on the route ofadministration. Physiologically and pharmaceutically acceptable carriersinclude diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials which are well known in the art. The term denotes anorganic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application. Thecomponents of the pharmaceutical compositions also are capable of beingco-mingled with the GHRH antagonist, and with each other, in a mannersuch that there is no interaction which would substantially impair thedesired pharmaceutical efficacy.

As used herein, the term “pharmaceutically acceptable carrier” refers tosolvents, dispersion media, coatings, antibacterial, isotonic andabsorption delaying agents, buffers, excipients, binders, lubricants,gels, surfactants that can be used as media for a pharmaceuticallyacceptable substance. The pharmaceutically acceptable carriers can belipid-based or a polymer-based colloid. Examples of colloids includeliposomes, hydrogels, microparticles, nanoparticles and micelles. Thecompositions can be formulated for administration by any of a variety ofroutes of administration, and can include one or more physiologicallyacceptable excipients, which can vary depending on the route ofadministration. Any of the growth hormone releasing hormone peptidesdescribed herein can be administered in the form of a pharmaceuticalcomposition. The growth hormone releasing hormone peptides can beadministered by any conventional route, including injection or bygradual infusion over time. The administration may, for example, beoral, intravenous, intratumoral, intraperitoneal, intramuscular,intracavity, subcutaneous, inhalation, or transdermal.

As used herein, the term “excipient” means any compound or substance,including those that can also be referred to as “carriers” or“diluents.” Preparing pharmaceutical and physiologically acceptablecompositions is considered routine in the art, and thus, one of ordinaryskill in the art can consult numerous authorities for guidance ifneeded. The compositions can also include additional agents (e.g.,preservatives).

The pharmaceutical compositions as disclosed herein can be prepared fororal or parenteral administration. Pharmaceutical compositions preparedfor parenteral administration include those prepared for intravenous (orintra-arterial), intramuscular, subcutaneous, intrathecal orintraperitoneal administration. Paternal administration can be in theform of a single bolus dose, or may be, for example, by a continuouspump. In some aspects, the compositions can be prepared for parenteraladministration that includes dissolving or suspending the growth hormonereleasing hormone peptides in an acceptable carrier, including but notlimited to an aqueous carrier, such as water, buffered water, saline,buffered saline (e.g., PBS), and the like. One or more of the excipientsincluded can help approximate physiological conditions, such as pHadjusting and buffering agents, tonicity adjusting agents, wettingagents, detergents, and the like. Where the compositions include a solidcomponent (as they may for oral administration), one or more of theexcipients can act as a binder or filler (e.g., for the formulation of atablet, a capsule, and the like). Where the compositions are formulatedfor application to the skin or to a mucosal surface, one or more of theexcipients can be a solvent or emulsifier for the formulation of acream, an ointment, and the like.

In some aspects, the compositions disclosed herein can be formulated fororal, intramuscular, intravenous, intratracheal, subcutaneous orintraperitoneal administration.

In some aspects, the compositions disclosed herein can be administeredby injection or by gradual infusion over time. In some aspects, theadministration can be oral, intramuscular, intravenous, intratracheal,subcutaneous or intraperitoneal, intratumoral, intracavity,subcutaneous, or transdermal.

The pharmaceutical compositions can be sterile and sterilized byconventional sterilization techniques or sterile filtered. Aqueoussolutions can be packaged for use as is, or lyophilized, the lyophilizedpreparation, which is encompassed by the present disclosure, can becombined with a sterile aqueous carrier prior to administration. The pHof the pharmaceutical compositions typically will be between 3 and 11(e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7and 8). The resulting compositions in solid form can be packaged inmultiple single dose units, each containing a fixed amount of theabove-mentioned agent or agents, such as in a sealed package of tabletsor capsules. The composition in solid form can also be packaged in acontainer for a flexible quantity, such as in a squeezable tube designedfor a topically applicable cream or ointment. The compositions can alsobe formulated as powders, elixirs, suspensions, emulsions, solutions,syrups, aerosols, lotions, creams, ointments, gels, suppositories,sterile injectable solutions and sterile packaged powders. The activeingredient can be any of the growth hormone releasing hormone peptidesdescribed herein in combination with one or more pharmaceuticallyacceptable carriers. As used herein “pharmaceutically acceptable” meansmolecules and compositions that do not produce or lead to an untowardreaction (i.e., adverse, negative or allergic reaction) whenadministered to a subject as intended (i.e., as appropriate).

Any of the GHRH antagonists (or a composition comprising any of the GHRHantagonists) can be administered in effective amounts. An “effectiveamount” with respect to a GHRH antagonist according to the teachingsherein is that amount of a GHRH antagonist composition that alone, ortogether with further doses, produces the desired response, e.g., treatscancer, decreases the proliferation of cancer cells, inhibits tumorgrowth, kills tumor cells, treats pulmonary fibrosis, reduces lunginflammation, reduces lung scarring, ameliorates one or more symptoms ofpulmonary fibrosis, or reduces expression of one or more T cell receptorcomplex genes. In the case of treating a cancer or treating pulmonaryfibrosis, a desired response can be inhibition of progression of thedisease. This may involve slowing the progression of the diseasetemporarily, although more preferably, it involves halting theprogression of the disease permanently. In some aspects, diseaseprogression and/or cancer cell death can be monitored by routinemethods. In some aspects, administration of the GHRH antagonist delaysonset or prevents the onset of cancer. In some aspects, administrationof the GHRH antagonist can mediate a reduction in tumor size, such as areduction in primary tumor volume, or halts or slows growth of a tumor.Optionally, the method described herein can reduce tumor size by atleast 1%, 3%, 5%, 10% or more. Alternatively or in addition, the methoddescribed herein reduces tumor burden (by, for example, 1%, 3%, 5%, 10%or more); slows, delays, or prevents metastasis; results in a reductionin cancer specific antigen levels (e.g., prostate specific antigenlevels) in the blood (by, for example, 10% or more); or improves cancergrading used by clinicians (e.g., Gleason score for prostate cancer). Insome aspects, the method described herein decreases cancer cellproliferation by at least 1%, 3%, 5%, 10% or more. In some aspects; themethod described herein reduces levels of prostate specific antigen(PSA) (by, for example, 10%, 15%, 20% or more) in the blood of thesubject receiving treatment. In some aspects, the method describedherein reduces level of PSA by 2-fold, (5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold,80-fold or more) in the blood of the subject receiving treatment. Itwill be appreciated that “treating cancer” does not require a completeelimination of the disease; any improvement in the disease state isbeneficial to the patient and contemplated herein. Additionally, it willbe appreciated that “inhibiting tumor growth” does not require completearrest of tumor growth; slowing of tumor growth or progression iscontemplated.

Amounts of GHRH antagonist will depend on the severity of the condition,the individual patient parameters including age, physical condition,size and weight, the duration of the treatment, the nature of concurrenttherapy (if any), the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. It isgenerally preferred that a maximum dose of the individual components orcombinations thereof be used, that is, the highest safe dose accordingto sound medical judgment. It will be understood by those of ordinaryskill in the art, however, that a patient may insist upon a lower doseor tolerable dose for medical reasons, psychological reasons or forvirtually any other reasons.

The doses of growth hormone releasing hormone peptides administered to asubject can be chosen in accordance with different parameters, such asthe mode of administration used. In the event that a response in asubject is insufficient at the initial doses applied, higher doses (oreffectively higher doses by a different, more localized delivery route)may be employed to the extent that patient tolerance permits.

In general, doses of growth hormone releasing hormone peptides can beformulated and administered in doses between 0.5 mg/kg to about 500mg/kg. In some aspects, the growth hormone releasing hormone peptidescan be formulated and administered at a dose ranging from 0.5 mg/kg toabout 5 mg/kg, 0.5 mg/kg to about 200 mg/kg, or about 1 mg/kg to about100 mg/kg, or about 1 mg/kg to about 50 mg/kg. In some aspects, thegrowth hormone releasing hormone peptides can be formulated andadministered at a dose of about 0.5 mg/kg or about 1 mg/kg, or about 5mg/kg, or about 10 mg/kg, or about 20 mg/kg, or about 30 mg/kg, or about40 mg/kg, or about 50 mg/kg, or about 60 mg/kg, or about 70 mg/kg, orabout 80 mg/kg, or about 90 mg/kg, or about 100 mg/kg.

In some aspects, doses of growth hormone releasing hormone peptides canbe formulated and administered in a dose of ranging from about 30 mg toabout 300 mg (or about 30 mg to about 50 mg, or about 30 mg to about 100mg, or about 50 mg to about 150 mg, or about 75 mg to about 200 mg, orabout 100 mg to about 300 mg). In some aspects, the growth hormonereleasing hormone peptides can be formulated and administered at a doseof about 30 mg, or about 35 mg, or about 40 mg, or about 45 mg, or about50 mg, or about 55 mg, or about 60 mg, or about 65 mg, or about 70 mg,or about 75 mg, or about 80 mg, or about 85 mg, or about 90 mg, or about100 mg, or about 150 mg, or about 200 mg, or about 250 mg or about 300mg).

In some aspects, administration of GHRH antagonist compositions tomammals other than humans, e.g., for testing purposes or veterinarytherapeutic purposes, can be carried out under substantially the sameconditions as described above.

EXAMPLES Example 1: Synthesis of GHRH Antagonists

A plurality of AVR growth hormone-releasing hormone (GHRH) antagonistswere synthesized using Fmoc-chemistry. The resulting GHRH antagonistscontained modifications at positions 0, 6, 8, 10, 11, 12, 20, 21, 29 and30 compared to a reference set of GHRH antagonists (“MIA” peptides). SeeTables 1 and 2 below.

C-terminal methylamide or ethylamide AVR-GHRH antagonists weresynthesized using the Fmoc peptide synthesis on[3-((Methyl-Fmoc-amino)-methyl)indol-1-yl]acetyl AM resin or[3-{ethyl-Fmoc-amino)-methyl)indol-1-yl]acetyl AM resin. Before startingthe synthesis, the Fmoc group was removed from the resin with 20%piperidine in DMF for 20 min. The side chain of Fmoc-amino acids wereprotected with acid unstable groups such as β-tert-Butyl ester for ASP,tert-Butyl(But) for Ser, Thr and Tyr;Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Har, DArg, Arg;N8-tert-Butoxycarbonyl (Boc) for Orn; Nγ-trityl for Asn; Nδ-trityl forGln. Nε-trityl for Lys and Nim-trityl for His; The coupling wasperformed by using 3 equivalents of Fmoc amino acid and[2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate](HBTU) mixed in the 0.5 M 1-Hydroxybenzotriazole(HOBT) DMF solution, followed by addition of 6 equivalents ofN,N-Diisopropylethylamine (DIPEA) and stirred for a few minutes tobecome a complete solution, then immediately added to the resin andshaken for 1-2 hours to finish the coupling reaction. The resin withwashing and deprotection of Fmoc group was continued for next couplingwith following Fmoc acid.

Exemplary synthesis of AVR-235: The following Fmoc amino acid werecoupled in the indicated order on methylamide resin: Fmoc-Har(pbf),Fmoc-DArg(pbf), Fmoc-Nle, Fmoc-Ile, Fmoc-Asp(oBut), Fmoc-Gln(Trt),Fmoc-Leu, Fmoc-Leu, Fmoc-Lys(Boc), Fmoc-Arg(pbf), Fmoc-Ala,Fmoc-Ser(But), Fmoc-Leu, Fmoc-Gln(Trt), Fmoc-Abu, Fmoc-Leu, Fmoc-Val,Fmoc-Lys(Boc), Fmoc-Arg(pbf), Fmoc-Tyr(Me), Fmoc-Har(pbf), Fmoc-Ala,Fmoc-Thr(But), Fmoc-5FPhe, Fmoc-Ile, Fmoc-Ala, Fmoc-Asp(oBut),Fmoc-DArg(pbf), Fmoc-Tyr(But), Fmoc-Ada and 5FPhAc to obtain theprotected resin5FPhAc-Ada-Tyr(But)-DArg(pbf)-Asp(oBut)-Ala-Ile-5FPhe-Thr(But)-Ala-Har(pbf)-Tyr(Me)-Arg(pbf)-Lys(Boc)-Val-Leu-Abu-Gln(Trt)-Leu-Ser(But)-Ala-Arg(pbf)-Lys(Boc)-Leu-Leu-Gln(Trt)-Asp(oBut)-Ile-Nle-DArg(pbf)-Har(pbf)-NHCH₃-®. The protected peptide resin was treated with a mixed reagentand scavengers containing TFA/thioanisol/1,2-Ethanedithiol(EDT)/Anisol/H2O/Phenol (85%/5%/3%/2%/3%/2% by volume) at roomtemperature for 3 hr. The crude peptide was precipitated with tert-butylmethyl ether and purified with HPLC and analyzed by mass spectrometry.

The purification of the crude peptides was performed on a Beckman GoldHPLC System (Beckman Coulter, Inc., Brea, Calif.) equipped with 127Psolvent Module, model 166P UVVIS Detector, using an XBridge™ reversedphase column (10 mm×250 mm), packed with C18 silica gel, 300A° poresize, 5 m particle size (Waters Co., Milford, Mass.). The peptides wereeluted with a solvent system consisting of solvent A (0.1% aqueous TFA)and solvent B (0.1% TFA in 70% aqueous acetonitrile (MeCN)) in a lineargradient mode of 30-55% of solvent B for 120 min at a flow rate of 5ml/min. The eluent was monitored at 220 and 280 nm, and the fractionswere examined by analytical HPLC using a Hewlett-Packard Model HP-1090liquid chromatograph and pooled to give maximum purity. Analytical HPLCwas carried out on a Supelco Discovery HS C18 reversed-phase column (2.1mm×50 mm, C18, 300A° pore size, 3 m particle size; Supelco Bellefonte,Pa.) using gradient elution from 40 to 80% B for 40 min with a solventsystem consisting of solvents A and B, defined above, with a flow rateof 0.2 ml/min. The peaks were monitored at 220 and 280 nm. The peptideswere judged to be substantially (>95%) pure by analytical HPLC.Molecular masses were determined by Agilent 6210 time-of-flight massspectrometry in conjugation with 1200 CapLC (Agilent Technologies 6210Time-of Light LC/MS, Santa Clara, Calif.). Peptides were eluted on anAgilent Zorbax C18 column (0.5 mm×150 mm, 300A° pore size, 5 m particlesize, Agilent, Santa Clara, Calif.) at a flow rate of 15 l/min with alinear gradient from 35 to 85% B for 30 min. Solvent A is 0.1% formicacid (FA), Solvent B is 90% aqueous MeCN/0.1% FA. TOF settings are asfollow: capillary voltage: 4000V; drying gas flow: 7 L/min; drying gastemperature: 300° C.; nebulizer gas: 30 psi; fragmentor voltage: 350V.

Exemplary synthesis of AVR-354: The following Fmoc amino acid werecoupled in indicated order on the[3-((Methyl-Fmoc-amino)-methyl)indol-1-yl]acetyl AM resin Fmoc-Ada,Fmoc-Har(pbf), Fmoc-DArg(pbf), Fmoc-Nle, Fmoc-Ile, Fmoc-Asp(oBut),Fmoc-Gln(Trt), Fmoc-Leu, Fmoc-Leu, Fmoc-Lys(Boc), Fmoc-Arg(pbf),Fmoc-Ala, Fmoc-Ser(But), Fmoc-Leu, Fmoc-Gln(Trt), Fmoc-Abu, Fmoc-Leu,Fmoc-Val, Fmoc-Lys(Boc), Fmoc-Arg(pbf), Fmoc-5FPhe, Fmoc-Har(pbf),Fmoc-Ala, Fmoc-Thr(But), Fmoc-Cpa, Fmoc-Fmoc-Ala, Fmoc-Asp(oBut),Fmoc-DArg(pbf), Fmoc-Tyr(But), Fmoc-Ada and 5FPhAc.

Synthesis of C-terminal amide compounds of AVR 333, AVR 352 and AVR353:AVR-333, AVR-352 and AVR-353 were synthesized on Rink amide MBHA resinwith Fmoc synthesis procedure as described in the synthesis of AVR-235.

For the synthesis of AVR-333, the following Fmoc amid acids were coupledin indicated order on the resin: Fmoc-Ada, Fmoc-Har(pbf),Fmoc-DArg(pbf), Fmoc-Nle, Fmoc-Ile, Fmoc-Asp(oBut), Fmoc-Gln(Trt),Fmoc-Leu, Fmoc-Leu, Fmoc-Lys(Boc), Fmoc-Arg(pbf), Fmoc-Ala,Fmoc-Ser(But), Fmoc-Leu, Fmoc-Gln(Trt), Fmoc-Abu, Fmoc-Leu, Fmoc-Val,Fmoc-Lys(Boc), Fmoc-Arg(pbf), Fmoc-Tyr(Me), Fmoc-Har(pbf),Fmoc-Asn(Trt), Fmoc-Thr(But), Fmoc-Cpa, Fmoc-Ile, Fmoc-Ala,Fmoc-Asp(oBut), Fmoc-DArg(pbf), Fmoc-Tyr(But), Fmoc-Ada and 5FPhAc.

For the synthesis of AVR 352, the following Fmoc amino acids werecoupled in indicated order on Rink amide MBHA resin: Fmoc-Ada,Fmoc-Har(pbf), Fmoc-DArg(pbf), Fmoc-Nle, Fmoc-Ile, Fmoc-Asp(oBut),Fmoc-Gln(Trt), Fmoc-Leu, Fmoc-Leu, Fmoc-Lys(Boc), Fmoc-Arg(pbf),Fmoc-Ala, Fmoc-Ser(But), Fmoc-Leu, Fmoc-Gln(Trt), Fmoc-Abu, Fmoc-Leu,Fmoc-Val, Fmoc-Lys(Boc), Fmoc-Arg(pbf), Fmoc-5FPhe, Fmoc-Har(pbf),Fmoc-Ala, Fmoc-Thr(But), Fmoc-5FPhe, Fmoc-Ile, Fmoc-Ala, Fmoc-Asp(oBut),Fmoc-DArg(pbf), Fmoc-Tyr(But), Fmoc-Ada and 5FPhAc.

For the synthesis of AVR-353, the following Fmoc amino acids werecoupled in indicated order on Rink amide MBHA resin: Fmoc-Ada,Fmoc-Har(pbf), Fmoc-DArg(pbf), Fmoc-Nle, Fmoc-Ile, Fmoc-Asp(oBut),Fmoc-Gln(Trt), Fmoc-Leu, Fmoc-Leu, Fmoc-Lys(Boc), Fmoc-Arg(pbf),Fmoc-Ala, Fmoc-Ser(But), Fmoc-Leu, Fmoc-Gln(Trt), Fmoc-Abu, Fmoc-Leu,Fmoc-Val, Fmoc-Lys(Boc), Fmoc-Arg(pbf), Fmoc-5FPhe, Fmoc-Har(pbf),Fmoc-Ala, Fmoc-Thr(But), Fmoc-Cpa, Fmoc-Ile, Fmoc-Ala, Fmoc-Asp(oBut),Fmoc-DArg(pbf), Fmoc-Tyr(But), Fmoc-Ada and 5FPhAc.

TABLE 1 Amino acid replacements in AVR - GHRH antagonists and MIA-602and MIA-690 peptides. 0 6 8 9 10 11 12 20 21 29 30 MW MW-TFA MIA-602PhAC-Ada 5FPhe Ala — — His Orn His Orn Har-NH₂ 3931 4843 MTA-690 — Cpa —— 5FPhe — — — — — 3934 4846 AVR-235 5FPhAC-Ada 5FPhe — — Tyr(Me) Arg LysArg Lys Har-NH₂ 4101 5013 AVR-333 — Cpa Asn — — — — — — Har Ada-NH₂ 42715183 AVR-352 — 5FPhe Ala — — — — — — Har Ada-NH₂ 4284 5196 AVR-353 — Cpa— — 5FPhe — — — — Har Ada-NH₂ 4287 5199 AVR-354 — — — — — — — — — HarAda-NHCH₃ 4301 5213

TABLE 2 Structures of AVR antagonists tested in vivo. 0 1 2 3 4 5 6 7 89 10 11 12 13 14 15 16 MIA-602 PhAC-Ada Tyr D-Arg Asp Ala Ile 5FPhe ThrAla Har Tyr(Me) His Orn Val Leu Abu Gln MIA-690 PhAC-Ada Tyr D-Arg AspAla Ile Cpa Thr Ala Har 5FPhe His Orn Val Leu Abu Gln AVR-104 PhAC-AdaTyr D-Arg Asp Ala Ile Cpa Thr Ala Arg Tyr(Me) Arg Lys Val Leu Abu GlnAVR107 PhAC-Ada Tyr D-Arg Asp Ala Ile Cpa Thr Ala Arg Tyr Arg Lys ValLeu Abu Gln AVR-116 PhAC-Ada Tyr D-Arg Asp Ala Ile Cpa Thr Ala Arg 5FPheArg Lys Val Leu Abu Gln AVR-120 D-Phe-Ada Tyr D-Arg Asp Ala Ile Cpa ThrAla Arg Tyr(Me) Arg Lys Val Leu Abu Gln AVR-201 PhAC-Ada Tyr D-Arg AspAla Ile Cpa Thr Ala Arg Amp Arg Lys Val Leu Abu Gln AVR-234 PhAC-Ada TyrD-Arg Asp Ala Ile Cpa Thr Asn Arg Tyr(Me) Arg Lys Val Leu Abu GlnAVR-235 5FPhAC-Ada Tyr D-Arg Asp Ala Ile 5FPhe Thr Ala Har Tyr(Me) ArgLys Val Leu Abu Gln AVR-321 PhAC-Ada Tyr D-Arg Asp Ala Ile Cpa Thr AsnHar Tyr(Me) Arg Lys Val Leu Abu Gln AVR-322 5FPhAC-Ada Tyr D-Arg Asp AlaIle Cpa Thr Asn Har Tyr(Me) Arg Lys Val Leu Abu Gln AVR-332 PhAC-Ada TyrD-Arg Asp Ala Ile Cpa Thr Asn Har Tyr(Me) Arg Lys Val Leu Abu GlnAVR-333 5FPhAC-Ada Tyr D-Arg Asp Ala Ile Cpa Thr Asn Har Tyr(Me) Arg LysVal Leu Abu Gln AVR-352 5FPhAC-Ada Tyr D-Arg Asp Ala Ile 5FPhe Thr AlaHar Tyr(Me) Arg Lys Val Leu Abu Gln AVR-353 5FPhAC-Ada Tyr D-Arg Asp AlaIle Cpa Thr Ala Har 5FPhe Arg Lys Val Leu Abu Gln AVR-354 5FPhAC-Ada TyrD-Arg Asp Ala Ile Cpa Thr Ala Har 5FPhe Arg Lys Val Leu Abu Gln AVR-5425FPhAC-Ada Tyr D-Arg Asp Ala Ile Cpa Thr Ala Har 5FPhe His Orn Val LeuAbu Gln AVR-543 5FPhAC-Ada Tyr D-Arg Asp Ala Ile Cpa Thr Ala Har 5FPheHis Orn Val Leu Abu Gln AVR-552 5FPhAC-Ada Tyr D-Arg Asp Ala Ile Cpa ThrAla Har 5FPhe His Orn Val Leu Abu Gln AVR-553 5FPhAC-Ada Tyr D-Arg AspAla Ile 5FPhe Thr Ala Har Tyr(Me) His Orn Val Leu Abu Gln AVR-6205FPhAC-Ada Tyr D-Arg Asp Ala Ile Cpa Thr Asn Arg Tyr(Me) Arg Lys Val LeuAbu Gln 17 18 19 20 21 22 23 24 25 26 27 28 29 30 MW MW-TFA MIA-602 LeuSer Ala His Orn Leu Leu Gln Asp Ile Nle D-Arg Har-NH₂ 3928 4840 MIA-690Leu Ser Ala His Orn Leu Leu Gln Asp Ile Nle D-Arg Har-NH₂ 3931 4843AVR-104 Leu Ser Ala His Orn Leu Leu Gln Asp Ile Nle D-Arg Har-NH₂ 38914803 AVR107 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Nle D-Arg Har-NH₂3909 4821 AVR-116 Leu Ser Ala His Orn Leu Leu Gln Asp Ile Nle D-ArgHar-NH₂ 3950 4862 AVR-120 Leu Ser Ala His Orn Leu Leu Gln Asp Ile NleD-Arg Har-NH₂ 3919 4831 AVR-201 Leu Ser Ala His Orn Leu Leu Gln Asp IleNle D-Arg Har-NHCH₃ 3889 4801 AVR-234 Leu Ser Ala Arg Lys Leu Leu GlnAsp Ile Nle D-Arg Har-NHCH₃ 3981 4893 AVR-235 Leu Ser Ala Arg Lys LeuLeu Gln Asp Ile Nle D-Arg Har-NHCH₃ 4098 5010 AVR-321 Leu Ser Ala ArgLys Leu Leu Gln Asp Ile Nle D-Arg Har Aoc-NHCH₃ 4136 5048 AVR-322 LeuSer Ala Arg Lys Leu Leu Gln Asp Ile Nle D-Arg Har Aoc-NHCH₃ 4226 5138AVR-332 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Nle D-Arg Har Ada-NH₂4178 5090 AVR-333 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Nle D-Arg HarAda-NH₂ 4268 5180 AVR-352 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile NleD-Arg Har Ada-NH₂ 4281 5193 AVR-353 Leu Ser Ala Arg Lys Leu Leu Gln AspIle Nle D-Arg Har Ada-NH₂ 4284 5196 AVR-354 Leu Ser Ala Arg Lys Leu LeuGln Asp Ile Nle D-Arg Har Ada-NHCH₃ 4298 5210 AVR-542 Leu Ser Ala HisOrn Leu Leu Gln Asp Ile Nle D-Arg Har-NHCH₃ 4038 4950 AVR-543 Leu SerAla His Orn Leu Leu Gln Asp Ile Nle D-Arg Har Ada-NHCH₃ 4232 5144AVR-552 Leu Ser Ala His Orn Leu Leu Gln Asp Ile Nle D-Arg Har Ada-NH₂4218 5130 AVR-553 Leu Ser Ala His Orn Leu Leu Gln Asp Ile Nle D-Arg HarAda-NH₂ 4215 5127 AVR-620 Leu Ser Ala His Orn Leu Leu Gln Asp Ile NleD-Arg Har Ada-NH₂ 4224 5136

As shown in Table 1, certain AVR compounds have 5PhAc at N-terminal, Argat positions 2 and 20, Lys at positions 12 and 21, and modifiedC-terminal NH₂ with —NHCH₃ (AVR-235), Ada-NH₂ (AVR-253 and AVR-252), or-Ada-NHCH₃ (AVR-254).

Example 2: Evaluation of Inhibitory Effects of GHRH Antagonists In Vitro

The inhibitory effects of the various synthesized GHRH antagonists weretested in cell proliferation assays in various human cancer cell linesincluding stomach cancer (KATOIII, N87), colon cancer (HT-29),urothelial cancer (J82, RT4), pancreatic cancer (PANC-1, CFPAC-1),prostatic cancer (PC3), breast cancer (MX-1, HCC1806), ovarian cancer(SK-OV-3, OVCAR-3) and lung cancer (HCC827, H460).

Cell culture. Human cancer cell lines including pancreatic (PANC-1 andCFPAC-1), lung (HCC827 and H460), stomach (NCI-N87 and KATOIII),urothelial (J82, RT4), prostatic (PC3), breast (MX-1 and HCC 1806),colorectal (HT-29), and ovarian (SK-OV-3 and OVCAR-3) were obtained fromthe American Type Culture Collection (ATCC) and cultured at 37° C. in ahumidified 95% air/5% CO2 atmosphere in the medium recommended by ATCC.

In vitro cell viability. Cell viability was analyzed as follows: cancercells including pancreatic (PANC-1 and CFPAC-1), lung (HCC827 and H460),stomach (NCI-N87 and KATOIII), urothelial (J82, RT4), prostatic (PC3)and breast (MX-1 and HCC 1806), colorectal (HT-29) and ovarian (SK-OV-3and OVCAR-3) were grown on 96 well plates to ˜60% confluence, the cellswere starved for 24 h in culture medium containing 0.5% FBS. Cells werethen treated with GHRH antagonists MIA602 AVR compounds atconcentrations of 1, 2.5 or 5 μM, or vehicle (0.1% DMSO) inquintuplicates for 72 hours. The medium was replaced every 48 hours.Cell viability was measured using CellTiter 96 aqueous one solution kit(Promega). Inhibitory effects of tested AVR compounds in comparison tothat of MIA602 are presented as inhibitory potency.

Inhibitory effects of selected AVR compounds in comparison to that ofMIA602 are presented as inhibitory potency (e.g., percentage ofinhibition). Results for lung cancer cells HCC827 are shown in FIG. 2.FIG. 2 provides a summary of in vitro inhibition of cell viability bythe treatment of MIA-602 and selected AVR-antagonists at concentrationof 1, 2 and 5 μM for 72 hr. The cell lines tested including lung cancerHCC827 (FIG. 2A), pancreatic cancer CFPAC-1 (FIG. 2B), stomach cancerN87 (FIG. 2C), colon cancer HT-29 (FIG. 2D), breast cancers MX-1 andHCC1806 (FIGS. 2 E, F). Based on the results of cell proliferation assayafter cancer cells were treated with antagonist at concentration of 5 μMfor 72H, the relative inhibitory potency of AVR antagonists incomparison to MIA-602 is summarized in Table 3 A and B. Over 60 AVRcompounds were tested in 12 cancer cell lines.

TABLE 3A GHRH antagonists tested in cancer cell lines. GHRH AntagonistsStomach Lung Pancreatic Urothelial Prostatic Breast Groups N87 KATOIIIIICC827 CFPAC-1 Panc-1 J82 RT4 PC3 IICC1806 MX-1 MIA MIA602 1 1 1 1 1 11 1 1 1 AVR- 102 1.120 104 2.291 1.321 1.525 1.502 1.114 3.377 1.6971.440 1.220 1.310 105 1.090 1.120 1.380 0.640 1.030 1.160 107 1.2951.080 1.160 0.800 0.950 1.010 110 1.477 1.775 111 1.862 1.827 113 0.939115 1.963 1.652 1.399 1.049 2.552 116 2.484 1.202 2.470 1.445 1.2083.034 117 1.009 118 1.332 1.429 1.264 1.054 2.988 119 1.568 1.364 1202.339 1.271 1.423 1.617 1.306 4.841 1.611 AVR- 201 3.455 1.208 1.3011.047 1.322 0.984 1.924 0.933 1.045 202 1.065 1.200 1.010 0.920 2031.120 1.009 0.922 204 1.110 1.196 0.849 205 1.120 0.979 1.091 206 1.2001.128 0.980 1.175 207 1.138 0.901 1.219 208 0.945 0.903 0.966 209 1.1370.984 0.723 210 1.094 0.982 0.664 211 1.080 1.085 0.874 1.560 212 1.0111.111 1.119 213 1.112 1.111 1.071 214 0.979 0.967 0.920 215 1.413 1.0120.820 231 1.056 232 1.466 233 1.881 1.561 1.354 0.867 1.474 234 1.7094.510 2.205 235 2.911 1.220 1.506 1.399 1.139 1.518 237 1.145 238 1.603AVR- 239 0.982 240 1.386 241 1.117 242 1.026 243 2.890 1.438 1.340 1.0341.296 244 1.795 1.160 1.118 0.970 1.176 245 1.264 AVR- 304 0.631 3110.919 312 1.191 313 1.233 321 1.414 322 1.296 324 5.227 331 0.907 3321.643 AVR- 510 1.064 520 1.361 530 1.171 540 1.626 551 1.456 552 4.3761.57 542 2.649 1.312 AVR- 610 1.479 620 1.769 3.753 1.567

TABLE 3B In vitro relative inhibitory potency of AVR-compound incomparison to MIA-602 GHRH Antagonists HCC827 CFPAC-1 NCI-N87 HT-29 MX-1HCC-1806 SKOV-3 OVCAR-3 MIA-602 1 1 1 1    1    1    1    1    AVR-2351.08 1.497 1.67 0.968 — — — — AVR-333 1.177 2.288 1.4 — — — — — AVR-3522.01 1.576 1.809 1.197 2.793 4.85  1.15* 1.68* AVR-353 1.641 2.922 3.4041.222 2.001 9.396 1.04* 1.55* AVR-354 2.595 1.67 — 1.403 1.594 3.991 — —AVR-543 0.961 2.05 0.908 0.999 — — — — AVR-553 2.582 — 1.26 — — — — —AVR-540 1.121 2.281 1.158 — — — — —

Based on the results of cell proliferation assay after cancer cells weretreated with antagonist at concentration of 5 □M for 72H, * atconcentration of 2 □M for 72 H.

In vitro assessment of the antagonistic activity of GHRH analogs. Ratswere decapitated and pituitaries were collected in pre-warmed HBSS(Hanks' Balanced Salt Solution). Tissue was then cut into small piecesand digested in HBSS medium containing 3% BSA, 50 μg/ml gentamycin and0.5% collagenase for 50 minutes at 37° C. Cells were further dispersedby pipetting and passed through a 100 μm cell strainer with the additionof DMEM containing 50 μg/ml gentamycin. 10% horse serum and 2.5% FBS(growth medium). Cells were harvested at 475×g for 2 minutes,resuspended in fresh growth medium and seeded onto poly-D-lysine-coated24-well plates (7 pituitaries/plate). Cells were left to recover for 4days. Growth medium was then replaced with serum-free DMEM for 4 hoursand GHRH analogs (at concentration of 20 nM) were added in DMEMcontaining 0.1% BSA for 30 minutes. Cells were replenished with mediumcontaining the same concentration of the analogs and 1 nM GHRH(1-29)NH₂for 30 minutes. Medium from this step was collected, centrifuged at 800g for 3 minutes and GH concentration was determined by ELISA (ALPCODiagnostics, Mill Valley, Calif.) according to the manufacturer'sinstruction.

FIG. 3 shows the inhibitory effects of GHRH AVR-antagonists on therelease of GH from rat pituitary cells (in vitro) in comparison toantagonists MIA-602. The data is summarized in Table 3C. AVR-235,AVR-352, and AVR-353 showed higher inhibitory effects than MIA-602.

TABLE 3C Inhibitory effects of GHRH AVR-antagonists on the release of GHfrom rat pituitary cells (in vitro) in comparison to antagonistsMIA-602. GHRH Relative GH antagonists release p value Control 1.00 ±0.04 MIA- 602 0.78 ± 0.01 <0.05 AVR-235 0.60 ± 0.02 <0.01 AVR-333 0.88 ±0.03 ns AVR-352 0.72 ± 0.03 <0.05 AVR-353 0.65 ± 0.08 <0.05 AVR-354 0.90± 0.02 ns AVR-540 0.88 ± 0.02 <0.05 AVR-543 1.10 ± 0.01 ns AVR-553 0.82± 0.04  0.06

Example 3: Evaluation of Tumor Inhibitory Activity of GHRH AntagonistsIn Vivo

The inhibitory activity of the GHRH antagonists that displayed highinhibitory properties in the in vitro assays described in Example 2(i.e., the nineteen antagonists provided in Table 2) were assessed invivo as to their effect on the growth of human cancers xenografted intonude mice. The peptides' activity was compared with GHRH antagonistMIA-602. All statistical analyzses were performed from comparing GHRAantagonists to non-treated tumor (control).

Peptides and chemicals. GHRH antagonists MIA-602 and AVR-GHRHantagonists (see Table 2 above) were synthesized by solid phase methodsand purified by HPLC. The peptides were dissolved in DMSO and furtherdiluted in 10% 1,2-propanediol. The final concentration of peptides was50 □g/ml in 10% 1,2-propanediol containing 0.1% of DMSO.

Cell culture. Human cancer cell lines including pancreatic (PANC-1 andCFPAC-1), lung (HCC827 and H460), stomach (NCI-N87), prostate (PC3),brest (MX-1 and HCC-1806), colorectal (HT-29), and ovarian (SK-OV-3 andOVCAR-3) were obtained from the American Type Culture Collection (ATCC)and cultured at 37° C. in a humidified 95% air/5% CO2 atmosphere in themedium recommended by ATCC.

Xenograft models of human cancers in nude mice. Female athymic (NCrnu/nu) nude mice, 5- to 6-weeks-old, obtained from the Envigo labs(Tampa, Fla.), were housed in laminar airflow cabinets underpathogen-free conditions with a 12-h light/12-h dark schedule, and werefed autoclaved standard chow and water. To generate mice withxenografted tumors, tumor xenografts were initiated by subcutaneous(s.c.) injection of 107 human cancer cells into female nude mice. Afterabout 4 weeks the resulting tumors were dissected and collected afterremoving necrosis tissues. The tumors were then cut into particles withsize of 3 mm3 and transplanted s.c. by trocar needle into experimentalanimals. When tumors grew to the size of 50-60 mm3 (by volume), micewere randomized into groups and treated daily for 4-8 weeks bysubcutaneous administration of 5 μg of GHRH antagonists MIA-602 orAVR-compounds (2-10 μg as indicated), or vehicle solutions (0.1% DMSO in10% 1,2-propanediol) respectively. Tumor sizes were measured weekly.

Statistical analysis. One-way ANOVA followed by Bonferroni comparisonwere performed. Data are expressed as Mean±SEM. To determine statisticalsignificance between animal groups, two-tailed Student's t-test wasconducted. Differences were considered significant when p<0.05.

Results. The five AVR GHRH antagonists shown in Table 3 were shown tohave the highest inhibitory effects on tumor growth when compared toGHRH antagonist MTA-602. FIG. 1A-N represents in vivo inhibition oftumor growth during the treatment with GHRH antagonist MIA-602 andselected AVR antagonists in nude mice xenografted with various humantumors including pancreatic cancer PANC-1 (FIG. 1A) and CFPAC-1 (FIG.1I); lung cancer HCC827 (FIGS. 1B, C) and H460 (FIG. 1H); stomach cancerN87 (FIGS. 1D-F); colon cancer HT-29 (FIG. 1G); breast cancer MX-1 andHCC-1806 (FIGS. 1J, K); ovarian cancer SK-OV-3 and OVACAR-3 (FIGS. 1 L,M) and prostate cancer PC3 (FIG. 1N). The percentages of inhibition oftumor growth at the end of treatments are summarized in Table 4 (A-N).See also Table 5.

TABLE 4 Oncological in vivo tests on AYR GHRH antagonists. A PancreaticCa PANC-1 (7 weeks) Animal (n) Tumor Group Tumor (t) Growth % SEM Pvalue % Inhibition Control (0.1% DMSO) n = 10/t = 13  2670.6 624.0 —MIA602 (5 μg/day) n = 9/t = 10 1572.7 392.3 ns 41.1% AVR-333 (5 μg/day)n = 7/t = 10 1384.4 169.0 0.09 48.2% AVR-352 (2.5 μg/day) n = 10/t = 12 2073.0 419.9 ns 22.4% AVR-352 (5 μg/day) n = 9/t = 10 1456.9 217.7 ns45.4% AVR-353 (2.5 μg/day) n = 8/t = 11 1482.8 216.4 ns 44.5% AVR-353 (5μg/day) n = 9/t = 13 1257.3 268.4 0.04 52.9% AVR-353 (10 μg/day) n = 9/t= 11 1005.1 203.9 0.03 62.4% B Lung Ca HCC827 (7 weeks) Animal (n) TumorGroup Tumor (t) Growth % SEM P value % Inhibition Control (0.1% DMSO) n= 10/t = 16 1311.5 161.1 — MIA602 (5 μg/day) n = 10/t = 15 662.2 88.60.047 49.5% AVR-235 (5 μg/day) n = 10/t = 15 771.0 178.0 ns 41.2%AVR-333 (5 μg/day) n = 10/t = 14 407.5 60.4 0.003 68.9% AVR-352 (5μg/day) n = 10/t = 13 371.7 102.0 0.003 71.6% AVR-353 (5 μg/day) n =10/t = 12 380.3 86.8 0.004 71.0% AVR-553 (5 μg/day) n = 10/t = 13 1132.0218.0 ns 13.7% AVR-543 (5 μg/day)  n = 9/t = 15 806.0 213.0 ns 38.6% CLung Ca HCC827 (8 weeks) Animal (n) Tumor Group Tumor (t) Growth % SEM Pvalue % Inhibition Control (0.1% DMSO) n = 11/t = 18 840.73 100.39 — —M1A602 (5 μg/day) n = 11/t = 18 523.03 142.47 0.04 37.8% AVR-353 (2.5μg/day) n = 12/t = 17 545.65 75.25 0.03 35.1% AVR-353 (5 μg/day) n =12/t = 17 420.79 54.94 0.001 50.0% AVR-353 (10 μg/day) n = 11/t = 15359.16 72.65 0.001 57.3% D Stomach Ca N87 (10 weeks) Animal (n) TumorSize Group Tumor (t) (mm³) SEM P value % Inhibition Control (0.1% DMSO)n = 6/t = 11 1224.00 486.30 — — MIA602 (2 μg/day) n = 6/t = 10 1065.00502.10 ns 13.0% MIA602 (5 μg/day) n = 6/t = 12 767.00 111.40 ns 37.3%AVR-235 (2 μg/day) n = 6/t = 12 558.60 143.10 ns 54.4% AVR-333 (2μg/day) n = 6/t = 12 454.00 58.30 ns 62.9% AVR-540 (2 μg/day) n = 6/t =12 657.80 167.60 ns 46.3% E Stomach Ca N87 (4 weeks) Animal (n) TumorGroup Tumor (t) Growth % SEM P value % Inhibition Control (0.1% DMSO) n= 6/t = 12 892.30 193.30 — — MIA602 (5 μg/day) n = 6/t = 12 427.20 72.100.035 52.1% AVR-543 (2 μg/day) n = 6/t = 12 406.8 100.2 0.061 48.4%AVR-543 (5 μg/day) n = 6/t = 12 313.90 39.6 0.008 64.8% AVR-553 (2μg/day) n = 6/t = 12 532.7 57.1 0.088 40.3% AVR-553 (5 μg/day) n = 6/t =12 283.3 42.0 0.006 68.3% F Stomach Ca N87 (4 weeks) Animal (n) TumorGroup Tumor (t) Growth % SEM P value % Inhibition Control (0.1% DMSO) n= 6/t = 12 892.30 193.30 — — MIA602 (5 μg/day) n = 6/t = 12 427.20 72.100.035 52.1% AVR-235 (2 μg/day) n = 6/t = 12 312.80 40.90 0.008 64.9%AVR-353 (2 μg/day) n = 6/t = 12 433.70 32.22 0.029 51.4% AVR-353 (5μg/day) n = 6/t = 12 312.20 42.00 0.008 65.0% G Colon Ca HT-29 (6 weeks)Animal (n) Tumor Size Group Tumor (t) (mm³) SEM P value % InhibitionControl (0.1% DMSO) n = 10/t = 19 725.40 121.80 — — MIA602 (5 μg/day) n= 10/t = 20 582.10 145.60 ns 16.5% AVR-353 (2 μg/day) n = 10/t = 16549.30 128.80 ns 24.2% AVR-353 (5 μg/day) n = 10/t = 19 494.70 109.30 ns31.8% AVR-354 (5 μg/day) n = 10/t = 19 417.90 66.86 0.033 42.3% H LungCa H460 (4 weeks) Animal (n) Tumor Group Tumor (t) Growth % SEM P value% Inhibition Control (0.1% DMSO) n = 11/t = 18 3382.6 236.4 MIA602 (5μg/day)  n = 9/t = 16 1602.7 163.5 <0.001 52.6% AVR-352 (5 μg/day) n =11/t = 18 1738.3 306.6 <0.001 48.6% AVR-353 (5 μg/day) n = 10/t = 161335.0 252.0 <0.001 60.5% I Pancreatic Ca CFPAC-1 (7 weeks) Animal (n)Tumor Group Tumor (t) Growth % SEM P value % Inhibition Control (0.1%DMSO) n = 8/t = 13 1970.8 312.8 — — MIA602 (5 μg/day) n = 7/t = 9 1587.5 373.9 ns 19.5% AVR-352 (2.5 μg/day) n = 8/t = 14 1616.9 166.1 ns18.0% AVR-352 (5 μg/day) n = 11/t = 18  1448.4 193.1 ns 26.5% AVR-353(2.5 μg/day) n = 8/t = 14 1294.1 269.1 ns 34.3% AVR-353 (5 μg/day) n =9/t = 11 1077.6 161.4 0.018 46.2% J Breast Ca MX-1 (4 weeks) Animal (n)Tumor Group Tumor (t) Growth % SEM P value % Inhibition Control (0.1%DMSO) n = 10/t = 20 1118.4 165.5 MIA602 (5 μg/day) n = 10/t = 20 677.3119.3 0.009 39.2% AVR-352 (5 μg/day) n = 10/t = 20 673.2 108.1 0.04139.5% AVR-353 (5 μg/day) n = 10/t = 20 774.2 110.1 0.046 30.5% AVR-354(5 μg/day) n = 10/t = 20 488.3 45.7 0.002 56.2% K Breast Ca HCC-1806 (5weeks) Animal (n) Tumor Group Tumor (t) Growth % SEM P value %Inhibition Control (0.1% DMSO) n = 10/t = 20 1194.7 280.1 MIA602 (5μg/day) n = 10/t = 20 529.4 63.1 0.041 55.7% AVR-352 (5 μg/day) n = 10/t= 20 522.7 116.2 0.056 56.2% AVR-354 (5 μg/day) n = 10/t = 20 680.2 92.9ns 43.1% L Ovarian Ca SK-OV-3 (7weeks) Animal (n) Tumor Size Group Tumor(t) (mm3) SEM P value % Inhibition Control (0.1% DMSO) n = 10/t = 201305.7 153.1 MIA602 (5 μg/day) n = 10/t = 20 810.1 68.1 0.038 38.0%AVR-352 (2.5 μg/day) n = 10/t = 19 718.4 135.6 0.009 45.0% AVR-352 (5μg/day) n = 10/t = 20 422.3 59.5 0.005 67.7% AVR-353 (2.5 μg/day) n =10/t = 19 1056.0 104.4 ns 19.1% AVR-353 (5 μg/day) n = 10/t = 20 910.7134.7 ns 30.9% M Ovarian Ca OVCAR-3 (9 weeks) Animal (n) Tumor SizeGroup Tumor (t) (mm3) SEM P value % Inhibition Control (0.1% DMSO) n =10/t = 20 480.2 43.9 MIA602 (5 μg/day) n = 10/t = 20 267.2 27.5 0.00344.4% AVR-352 (2.5 μg/day) n = 10/t = 20 307.7 43.6 0.021 35.9% AVR-352(5 μg/day) n = 10/t = 20 199.9 27.8 <0.001  58.4% AVR-353 (2.5 μg/day) n= 10/t = 20 357.0 24.8 ns 25.7% AVR-353 (5 μg/day) n = 10/t = 20 255.221.5 0.003 46.9% N Prostate Ca PC3 (8 weeks) Animal (n) Tumor Size GroupTumor (t) (mm3) SEM P value % Inhibition Control (0.1% DMSO) n = 10/t =20 826.7 78.6 MIA602 (5 μg/day) n = 10/t = 20 450.9 41.8 0.003 45.5%AVR-352 (5 μg/day) n = 10/t = 20 413.3 38.4 0.007 50.0% AVR-353 (5μg/day) n = 10/t = 20 496.7 49.9 0.008 39.9% AVR-354 (5 μg/day) n = 10/t= 19 343.6 28.8 0.002 58.4%

AVR GHRH antagonists demonstrating best inhibitiory activity compared toMIA-602 and MIA-690. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 MIA-602PhAC-Ada Tyr D-Arg Asp Ala Ile 5FPhe Thr Ala Har Tyr(Me) His Orn Val _euAbu Gln MIA-690 PhAC-Ada Tyr D-Arg Asp Ala Ile Cpa Thr Ala Har 5FPhe HisOrn Val _eu Abu Gln AVR-235 5FPhAC-Ada Tyr D-Arg Asp Ala Ile 5FPhe ThrAla Har Tyr(Me) Arg Lys Val _eu Abu Gln AVR-333 5FPhAC-Ada Tyr D-Arg AspAla Ile Cpa Thr Asn Har Tyr(Me) Arg Lys Val _eu Abu Gln AVR-3525FPhAC-Ada Tyr D-Arg Asp Ala Ile 5FPhe Thr Ala Har Tyr(Me) Arg Lys Val_eu Abu Gln AVR-353 5FPhAC-Ada Tyr D-Arg Asp Ala Ile Cpa Thr Ala Har5FPhe Arg Lys Val _eu Abu Gln AVR-354 5FPhAC-Ada Tyr D-Arg Asp Ala IleCpa Thr Ala Har 5FPhe Arg Lys Val _eu Abu Gln 17 16 19 20 21 22 23 24 2526 27 28 29 30 MW MW-TFA MIA-602 Leu Ser Ala His Orn Leu Leu Gln Asp IleNle D-Arg Har-NH₂ 3931 4843 MIA-690 Leu Ser Ala His Orn Leu Leu Gln AspIle Nle D-Arg Har-NH₂ 3934 4846 AVR-235 Leu Ser Ala Arg Lys Leu Leu GlnAsp Ile Nle D-Arg Har-NHCH₃ 4101 5013 AVR-333 Leu Ser Ala Arg Lys LeuLeu Gln Asp Ile Nle D-Arg Har Ada-NH₂ 4271 5183 AVR-352 Leu Ser Ala ArgLys Leu Leu Gln Asp Ile Nle D-Arg Har Ada-NH₂ 4284 5196 AVR-353 Leu SerAla Arg Lys Leu Leu Gln Asp Ile Nle D-Arg Har Ada-NH₂ 4287 5199 AVR-354Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Nle D-Arg Har Ada-NHCH₃ 43015213 Molecular weights are listed as free base and TFA salt. MIA-602(SEQ ID NO: 21); MIA-690 (SEQ ID NO: 22); AVR-235 (SEQ ID NO: 3);AVR-333 (SEQ ID NO: 4); AVR-352 (SEQ ID NO: 5); AVR-353 (SEQ ID NO: 6);and AVR-354 (SEQ ID NO: 7).

Example 4: Binding Affinities of AVR GHRH Antagonists

Receptor binding affinities in vitro. Preparation of human pituitarymembrane fractions and receptor binding of analogs of GH-RH wereperformed (Halmos G, Rekasi Z, Szoke B, Schally A V. (1993) Receptor3:87-97). Human pituitary was purchased from the National Hormone andPeptide Program. In the competitive binding analysis, 1251-labeled[His¹, Nle²⁷]-hGH-RH-(1-32)-NH₂ (0.2 nM) was displaced byGH-RH-antagonists at 10-6-10-12 M. The final binding affinities wereexpressed as IC₅₀ values and were calculated by using the LIGAND PCcomputerized curve-fitting program of Munson and Rodbard as modified byMcPherson (Halmos G, Rekasi Z, Szoke B, Schally A V. (1993) Receptor3:87-97). Relative affinities compared to hGH-RH(1-29)NH₂ werecalculated as the ratio of IC₅₀ (dose causing 50% inhibition of specificbinding to receptors) of the tested peptides to the IC₅₀ ofhGH-RH(1-29)NH₂. The values were calculated from technical duplicatetubes.

Nineteen new AVR analogs of GHRH were tested in the receptor bindingassay. Table 6 presents the IC₅₀ values of GHRH antagonists to membraneGHRH-receptors binding on human anterior pituitary cells. AVR-352,AVR-353, AVR-354, AVR-552 and AVR-553 showed the higher binding affinityin comparison to MIA-602.

TABLE 6 IC₅₀ values of new AVR hGH-RH analogs to membrane receptors onhuman anterior pituitary cells. Peptide SEQ ID No. Code IC₅₀ ^(a) (nM)22 MIA-690 0.53 21 MIA-602 0.27 8 AVR-104 0.59 9 AVR-107 0.61 10 AVR-1160.50 11 AVR-120 0.48 12 AVR-201 0.61 13 AVR-234 0.45 3 AVR-235 0.49 14AVR-321 0.23 15 AVR-322 0.25 23 AVR-332 0.34 4 AVR-333 0.30 5 AVR-3520.09 6 AVR-353 0.11 7 AVR-354 0.14 16 AVR-542 0.21 17 AVR-543 0.25 18AVR-552 0.17 19 AVR-553 0.15 20 AVR-620 0.31 ^(a) IC₅₀ values representmean of two to three determinations. (5FPhAc Ada Tyr DArg Asp Ala Ile5FPhe Thr Ala HArg Tyr(Me) Arg Lys Val Leu Abu Gln Leu Ser Ala Arg LysLeu Leu Gln Asp Ile Nle DArg HArg Ada NH₂; SEQ ID NO: 23; AVR-332).

Based on these studies GHRH analogs AVR-352, AVR-353, AVR-354, AVR-552and AVR-553 showed the highest binding affinities to the membranereceptors of human pituitary cells. And, in particular, AVR-352 andAVR-353 strong candidates for further development.

Example 5: Growth Hormone-Releasing Hormone Receptor AntagonistModulates Lung Inflammation and Fibrosis Due to Bleomycin

Abstract. Purpose. Growth hormone-releasing hormone (GHRH) is a 44-aminoacid peptide that regulates growth hormone (GH) secretion. It was testedwhether a GHRH receptor (GHRH-R) antagonist, MIA-602, would inhibitbleomycin-induced lung inflammation and/or fibrosis in C57Bl/6J mice.Methods. It was tested whether MIA-602 (5 μg or vehicle givensubcutaneously [SC] on days 1-21) would decrease lung inflammation (atday 14) and/or fibrosis (at day 28) in mice treated with intraperitoneal(IP) bleomycin (0.8 units on days 1, 3, 7, 10, 14 and 21). Bleomycinresulted in inflammation and fibrosis around airways and vessels evidenthistologically at days 14 and 28. Results. Inflammation (histopathologicscores assessed blindly) was less evident in mice treated with MIA-602for 14 days. After 28 days, lung hydroxyproline (HP) content increasedsignificantly in mice treated with vehicle; in contrast, lung HP did notincrease significantly compared to naïve controls in mice treated withGHRH-R antagonist. GHRH-R antagonist increased basal and maximal oxygenconsumption of cultured lung fibroblasts. Multiple genes related tochemotaxis, IL-1; chemokines, regulation of inflammation andextracellular signal-regulated kinases (ERK) were upregulated in lungsof mice treated with bleomycin and MIA-602. MIA-602 also prominentlysuppressed multiple genes related to the cellular immune responseincluding those for T cell differentiation, receptor signaling,activation, and cytokine production. Conclusions. MIA-602 reduced lunginflammation and fibrosis due to bleomycin. Multiple genes related toimmune response and T cell functions were downregulated, supporting theview that MIA-602 can modulate the cellular immune response to bleomycinlung injury.

Methods. Materials. The GHRH-R antagonist, MIA-602 (MW 4843), wassynthesized (PhAC-Ada, Tyr D-Arg, Asp, Ala, Ile, 5FPhe, Thr, Ala, Har,Tyr(Me), His, Orn, Val, Leu, Abu, Gin, Leu, Ser, Ala, His, Om, Leu, Leu,Gin, Asp, Ile; Nle, D-Arg, Har NH₂; SEQ ID NO: 21) at the Miami VAHS bysolid phase methods and purified by HPLC.

Experimental animals. 8-week old C57Bl/6J male mice (The JacksonLaboratory, Bar Harbor, Me.) were used in these experiments. Miceweighed about 26 grams at the start. Mice were randomly allocated to theexperimental groups and housed in identical filter top cages in aventilated rack. Mice were exposed to a 12-hour light/dark cycle and hadfree access to standard laboratory chow and water.

Bleomycin and GHRH-R antagonist treatment. Mice were treated with 0.8units of bleomycin (Bleomycin for Injection USP, Hospira, Lake Forest,Ill.) intraperitoneally (IP) on days 1, 3, 7, 10, 14 and 21 (Collins S,Chan-Li Y, Oh M, Vigeland C, Limjunyawong N, Mitzner W, Powell J D,Horton M R (2016). JCI Insight 1(4):1-13). Mice (randomly assigned) weresimultaneously treated subcutaneously (sc) with MIA-602 (5 μg/day) orits vehicle (100 μL/day) [14]. MIA-602 was dissolved in DMSO (ACS grade;Sigma-Aldrich) and diluted 1:500 in normal saline for daily sc injectionon days 0-21.

Tissue preparations. Mice were killed by CO2 inhalation before anytreatment, or on days 14 or 28 for micro-CT scans of lungs andharvesting of lung tissue (Vande Vel de G, Poelmans J, De Langhe E,Hillen A, Vanoirbeek J, Himmelreich U, Lories R (2016). Dis Model Mech9:91). The right mainstem bronchus was ligated and the right lungremoved and frozen at −80° C. for hydroxyproline (HP) assays. The leftlung was filled with 10% buffered formalin at −25 cm H₂O pressure, thebronchus ligated, and the lung fixed in formalin. Fixed lungs wereembedded in wax and 5 μm sections stained with hematoxylin and eosin(H&E) or Masson's trichrome stain.

Lung histopathology. Fibrosis was quantified in trichrome-stainedsections using a modification of the Ashcroft score that describesgrades of fibrosis. Inflammation in lung tissue was quantified in H&Estained sections (Hubner R, Gitter W, El Mokhtari N, Mathiak M, Both M,Bolte H, Freitag-Wolf S, Bewig B (2008). Biotechniques 44:507-517).

Hydroxyproline assay. Right lungs were weighed and homogenized in 10volumes of distilled water. Homogenates were hydrolyzed in 12 M HCl at120° C. for 3 hours (Kennedy J, Chandler D, Jackson R, Fulmer J (1986).Chest 89 (3 Suppl):123S-125S). Hydroxyproline was measuredcolorimetrically (560 nm) after hydrolysis using an assay kit (MAK008,Sigma-Aldrich, St. Louis, Mo.).

Micro-CT. Mice in each group were assessed by micro-CT scans (BrukerSkyScan 1176 Low Dose Micro-CT, Knotich, Belgium) of the lungs after CO2inhalation. A tracheostomy tube was inserted and lungs inflated withair. Scans were examined qualitatively to confirm development ofinfiltrates and reticular densities.

Lung fibroblasts. Newborn mouse lung fibroblasts (Mlg 2908) wereobtained from the American Type Culture Collection, Manassas, Va. andcultured in Eagle's minimal essential medium. Cells were grown in 6-wellplates before incubation with 1 or 5 μM MIA-602 or vehicle, RNAisolation, and oxygen consumption measurements.

Annexin V-propidium iodide assay. Apoptosis and necrosis were assessedwith annexin-V/propidium iodide staining (Annexin V: FITC Assay Kit, BioRad, Hercules, Calif. 94547) of lung fibroblasts incubated in 0, 1 or 5μM MIA-602 for 24 hours (Rieger A, Nelson K, Konowalchuk J, Barreda D.(2011). J Visualized Experiments 50:e2597). Culture medium andtrypsinized cells were collected and centrifuged at 400×g for 5 minutes.The pellet was resuspended in 100 μL annexin-V/propidium iodide. Thesuspension was incubated at 37° C. for 20 minutes, then washed with PBSand resuspended in 500 μL PBS and fluorescence quantified by BeckmanCoulter Life Sciences CytoFLEX benchtop flow cytometer (Beckman Coulter,Inc., Brea, Calif.).

RNA isolation. RNA was extracted from fixed lung tissue in paraffinblocks using a Quick-RNA™ FFPE Kit (R1008, Zymo Research, Irvine,Calif.) following the manufacturer's protocol (Patel P, Selvarajah S,Guerard K, Bartlett J, Lapointe J, Berman D, Okello J, Park P (2017).PLoS ONE 12:e0179732). Samples were deparaffinized, digested withproteinase K and decrosslinked at 65° C. for 15 minutes. RNA lysisbuffer was added and mixed with ethanol. The mixtures were transferredto spin columns to isolate total RNA.

RNA was extracted from cultured fibroblasts using a Direct-zol RNAMicroPrep kit (R2060; Zymo Research, Irvine, Calif.). Cells were washedwith PBS and lysed in TRIreagent™, then purified using Direct-zol RNAcolumns. DNase I treatment was done in columns and RNA eluted inDNase/Rnase-free water.

Cellular respiration. The effects of MIA-602 on mouse lung fibroblastoxygen consumption was measured using the Agilent Seahorse XF Cell MitoStress Test (Agilent Technologies, Santa Clara, Calif.) (Divakaruni A,Paradyse A, Ferrick D, Murphy A, Jastroch M (2014). Methods inEnzymology 547:309-354). Fibroblasts were incubated with vehicle, 1 or 5μM MIA-602 for 24 hours before measurement of oxygen consumption. Oneday before assay 80,000 fibroblasts were seeded into Seahorse 24-wellplates (n=6 wells per condition). Basal respiration was established andoligomycin, FCCP and rotenone plus antimycin A were added sequentiallyto measure ATP production, uncoupled respiration; and, non-mitochondrialoxygen consumption (Wangpaichitr, Wu C, Li Y, Nguyen D, Kandemir H, ShahS, Chen S, Feun L, Prince J, Kuo M, Savaraj N (2017). Oncotarget8(30):49275-49292).

RNAseq and Pathway analyses. At least 10 ng of total RNA was used asinput for the KAPA RNA HyperPrep Kit with RiboErase (HMR) to createribosomal RNA-depleted sequencing libraries, including sample indexing,to allow for multiplexing. Cluster generation and sequencing was done onthe Illumina cBOT and HiSeq 3000 using reagents provided by Illumina,finally generating >32 million single-end 100 base reads per sample.

De-multiplexed FASTQ files were created with Illumina supplied scriptsin BCL2FASTQ software (v2.17). Illumina adapters were trimmed using theTrim Galore! package and aligned to the mouse reference genome (mm10)with STAR aligner (v2.5.0a) with default alignment parameters (Dobin A,Davis C, Shlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M,Gingeras T (2013). Bioinformatics 29:15-21).

Gene counts were normalized using trimmed mean of M-values (TMM) method.Differential expression between groups was calculated with the exacttest implemented in edgeR (Robinson M, McCarthy D, Smyth G (2010).Bioinformatics 26:139-140).

Pathway enrichment analyses was completed using Enrichr online and DAVIDbioinformatics resource (Chen E, Tan C, Kou Y, Duan Q, Wang Z, MeirellesG, Clark N, Ma'ayan A (2013). BMC Bioinformatics 14:128; and Huang W.Sherman B, Lempicki R (2009) Systematic and integrative analysis oflarge gene lists using DAVID bioinformatics resources. Nature Protocols4:44-57).

Data analysis. Data are reported as arithmetic means±SEM or SD asindicated. Confidence intervals (5-95%) and ranges were used to describehistopathological scores. ANOVA followed by Dunnett's test or theBonnferoni correction was used for multiple comparisons with a controlor among groups (Kusuoka H, Hoffman J (2002). Circ Res 91:662-671). Atleast six mice were planned to be available at each time point in eachgroup, so that there would be sufficient power to detect a 20% change inlung hydroxyproline given an assumed coefficient of variance of 0.2.P≤0.05 was considered significant.

Results. Micro-CT scans. Mice treated with vehicle developed patchyinfiltrative densities that persisted to day 28. Mice that receivedbleomycin plus MIA-602, the GHRH-R antagonist, appeared to have lessprominent infiltrative densities in their lungs.

Lung hydroxyproline contents. Lung HP content did not increasesignificantly after 14 days of intermittent treatment with bleomycin inmice receiving MIA-602 or vehicle. However, after 28 days, lung HPcontent increased significantly in bleomycin-treated mice that receivedvehicle, but not in bleomycin-treated mice that received MIA-602 on days1-21. The data are summarized in FIG. 4.

Lung histopathology. After 14 days of intermittent bleomycin,inflammation and patchy, mild fibrosis were evident in mouse lungs. Morefibrosis and fewer inflammatory cells were evident after 28 days. Bothinflammatory changes and fibrosis were decreased in mice that receivedMIA-602 in addition to bleomycin. Representative examples are shown inFIG. 5.

MIA-602 appeared to reduce inflammation after 14 days in lungs ofbleomycin-treated mice (0.4-1.4 [5%-95% CI]; range 0.6-1.2; n=4),compared to mice receiving vehicle (0.6-2.1; 0.8-1.9; n=4). MIA-602appeared to reduce fibrosis after 28 days in lungs of bleomycin-treatedmice that received MIA-602 (1.1-2.9; 1.0-3.0; n=5), but not in micetreated with vehicle (1.9-2.8; 1.5-2.9; n=8).

Lung fibroblast response to MIA-602. Lung fibroblasts were exposed toMIA-602 or vehicle in vitro for 24 hours before annexin-V/PI assay.MIA-602 caused predominantly cytolytic cell death (1 μM, 9.3±1.1%; 5 μM,34.9±2.5%; P=0.0002) rather than apoptosis.

Cellular respiration. In vitro, MIA-602 at 5 μM concentration incomplete medium increased fibroblast basal respiration and maximalrespiration (after FCCP), compared to vehicle. Both 1 and 5 μM MIA-602also appeared to increase non-mitochondrial respiration (after antimycinA and rotenone). These data are summarized in FIG. 6.

RNA-seq gene expression. The effects of MIA-602 or vehicle was exploredon gene expression in lungs from mice treated in vivo with bleomycin for28 days (data not shown). Several physiologically relevant genes wereexpressed differently after treatment with bleomycin (absolute foldchange >1.5 and FDR<0.01). Specifically, after 28 days during whichbleomycin was administered, genes related to the extracellular matrix,Wnt regulation and signaling, and the extracellular region wereupregulated, consistent with known effects of bleomycin. Severalrelevant genes were found to be downregulated by bleomycin, includingthose related to lung morphogenesis and development, extracellularmatrix organization, and alveolar septal development.

Transcriptome profiles then showed numerous genes expressed differentlyafter treatment with MIA-602. Inversely modulated genes were highlyenriched in pathways related to the adaptive immune response, T celldifferentiation, T cell signaling, extracellular matrix organization, Tcell activation and differentiation and cytokine production, consistentwith the putative anti-inflammatory and anti-fibrotic effects ofMIA-602. The ten most differentially expressed genes detected in lungtissue treated with MIA-602 compared to vehicle treatment are shown inTable 7. Those genes differentially expressed in fibrotic lungs frombleomycin-exposed mice treated with MIA-602 compared to vehicle aredisplayed as a heat map in FIG. 7.

TABLE 7 Clusters of the differentially expressed genes and theirontology between bleomycin-induced pulmonary fibrosis treated vs.untreated with MIA-602. Adjusted Name Gene list P-value P-value T celldifferentiation CD4, CD8A, RAG2, 0.000003374 0.0007392 (GO:0030217) RAG1T cell activation (GO:0042110) CD4, CD8A, CD3G, 0.000003777 0.0007392CD3E, LAT T cell receptor signaling pathway PSMB11, CD4, 0.0000046300.0007392 (GO:0050852) THEMIS, CD3G, CD3E, LAT Antigen receptor-mediatedPSMB11, CD4, 0.00006007 0.007193 signaling pathway (G0:0050851) THEMIS,CD3E, LAT V(D) J recombination RAG2, RAG1 0.0001910 0.01829 (GO:0033151)Enzyme linked receptor protein CD4, CD8A, NPPA, 0.0002969 0.02370signaling pathway (GO:0007167) CD3A T cell differentiation in thymusRAG2, RAG1 0.0004471 0.02677 (GO:0033077) Regulation of leukocytecell-cell CD4, LAT 0.0004471 0.02677 adhesion (GO:1903037) Regulation oflymphocyte CD4. LAT 0.0006144 0.03270 activation (G0:0051249) Lymphocytedifferentiation CD4, RAG2, RAG1 0.0009974 0.04777 (GO:0030098)

Similarly, the in vitro effects of 1 and 5 μM MIA-602 was directlytested on normal mouse lung fibroblasts (not exposed to bleomycin) andfound significant downregulation of genes involved in collagen fibrilorganization, cell-matrix adhesion and elastic fiber assembly,consistent with the demonstrated anti-fibrotic effects of MIA-602.Upregulated fibroblast genes included those related to protein kinaseactivity, the JAK-STAT cascade, cell cycle and DNA replication includinghistones.

Discussion Pathophysiological GH secretion and IGF-1 activation havegrowth promoting effects in the lung resulting in increased alveolarsize (Garcia-Rio F, Pino J, Diez J, Ruiz A, Villasante C, Villamor J(2001). Am J Respir Crit Care Med 164:852-857). IGF-1 itself increasesα-smooth muscle actin in lung fibroblasts and promotes a myofibroblastphenotype. The pituitary type GHRH receptor is present in both normaland IPF lung tissue (Jackson R, Ai L, Zhang C, Zhang X, Delcroix G,Lazerson A, Mirsaeidi M, Schally A (2018). European Respiratory Journal52 (suppl 62): OA5349), suggesting that local secretion of GH may occurphysiologically and have direct effects on lung tissue.

MIA-602 partially inhibits both lung inflammation and fibrosis, assessedhistopathologically and biochemically, after intraperitoneal bleomycin.RNA-seq data, importantly, show suppression of the adaptive immuneresponse, T cell differentiation and activation and cytokine productionby MIA-602 in bleomycin-treated mouse lungs. The effects of the GHRH-Rantagonist observed have implications for fibrosing lung diseases inhumans, and they could, importantly, reveal novel pathways amenable toclinical drug development (Jenkins R, Moore B, Chambers R, Eickelberg O,Konigshoff M, Kolb M, Laurent G J, Nanthakumar C B, Olman M, Pardo A,Selman M, Sheppard D, Sime P, Tager A, Tatler A, Thannickal V, White E(2017). Am J Respir Cell Mol Biol 56:667-679).

Initially, development of lung infiltrates due to bleomycin wasconfirmed with micro CT scans, inflammation and fibrosis withhistopathological examination, and increased collagen with biochemicalassays. Similar models have been used successfully in the development ofantifibrotic drugs, including pirfenidone and nintedanib.

Senescent fibroblasts that display respiratory abnormalities, indicatingmitochondrial damage, express both STAT3 and p21 as markers of thesenescent phenotype (Waters D, Blokland K, Pathinayake P, Wei, SchuligaM, Jaffar J, Hansbro P, Prele C, Mutsaeres S, Bartlett N, Grainge C,Knight D (2019). Am J Resp Cell Molec Biol 10.1165/rcmb.2018-03280C).MIA-602 downregulates p21 activated kinase and STAT3 and NFκB in gastriccancer cells (Gan J, Ke X, Jiang J, Dong H, Yao Z, Lin Y, Lin W, Wu X,Yan S, Zhuang Y, Chu W, Cai R, Zhang X, Cheung H, Block N, Pang C,Schally A, Zhang H (2016). Proc Natl Acad Sci USA 113:14745-14750). GHRHantagonists like MIA-602 could modulate the senescent phenotype leadingto fibrosis, and conceivably be one of the mechanisms that lessens thefibrotic response in this model.

In vitro, MIA-602 at micromolar concentrations increased basal andmaximal mitochondrial respiration, and it resulted in marked cytolyticdeath of mouse lung fibroblasts. Mitochondrial dysfunction and loss ofapoptotic potential occur in fibroblasts from TPF lungs, and enhancementof mitochondrial function itself by MIA-602 at lower concentrations invivo might modulate fibrosis by maintaining the capacity for mitophagyand apoptosis (Ryter S, Rosas I, Owen C, Martinez F, Choi M, Lee C,Elias J, Choi A. (2018). Ann Am Thorac Soc 15(Suppl 4):5266-S272).

GHRH-R antagonists decrease lipid peroxidation, protein carbonyls andnitrotyrosine in prostate cancer cells (Rekasi Z, Varga J, Schally A,Halmos G, Armatis P, Groot K, Czompoly T (2001). Endocrinology141:2120-2128), indicating antioxidant effects that would augment theirother anti-inflammatory effects (Ren J, Yu Q, Ma D, Liang W, Leung P, NgT, Chu W, Schally A, Pang C, Chan S (2019). Exp Eye Res 181:277-284).Both GH and IGF-1 stimulate neutrophil superoxide (02-) production(Schally A, Varga J, Engel J (2007). Nature Clin Pract Endo Metab4:33-43). Since GHRH-R antagonists inhibit GH secretion and IGF-1activation (Fu Y, Arkins S, Wang B, Kelley K. J Immunol 146:1602-1608,1991), it could be predicted that they would inhibit both 02- andhydrogen peroxide (H₂O₂) release during the inflammatory phase of injury(Warwick-Davies J, Lowrie D, Cole P (1995). J Immunol 154:1909-1918).Cellular levels of oxidant stress are decreased by MIA-602, and itsantioxidant effect would limit redox signaling in response to receptorligation (Barbutis N, Schally A (2008). PNAS 105:20470-20475).

MIA-602 disrupts the PI3/AKT pathway in several experimental systems.PI3K/AKT signaling is involved in the pathogenesis of bleomycin-inducedfibrosis (Kral J, Kuttke M, Schrottmaier W, Bimecker B, Warszawska J,Wemig C, Paar H, Salzmann M, Sabin E, Brunner J S, Osterreicher C, KnappS, Assinger A, Schabbauer G (2016). Sci Rep 6:23034 doi:10.1038/srep23034), and suppression of the PI3K/AKT pathway by GHRHantagonist could also lessen lung fibrosis. GHRH clearly appearsinvolved in the lung's response to treatment with bleomycin andsubsequent healing.

Genes related to the extracellular matrix and the Wnt pathway were overexpressed in fibrotic lungs from mice treated with bleomycin, comparedgene expression in naïve controls (Cabrera S, Selman M, Lonzano-BolanosA, Konishi K, Richards T, Kaminski N, Pardo A (2013). Am J Physiol LungCell Molec Physiol 304:L593-L601). In contrast, genes related toepithelial tube branching, lung morphogenesis and lung development wereunder expressed after bleomycin and development of fibrosis. Genesrelated the immune response, cellular adhesion and remodeling, and Tcell signaling were also found to be upregulated in lungs from ratstreated with TGF-ß (Huang X, Li L, Ammor R, Zhang Y, Wang Y, Ravi K,Thompson J, Jarai G (2019). Am J Physiol Lung Cell Molec Physiol 316:L348-L357).

T cells play an important role in the lungs of patients with pulmonaryfibrosis (Simonian P, Roark C, Diaz del Valle F, Palmer B, Douglas I S,Ikuta K, Born W, O'Brien R, Fontenot A (2006). J Immunol 177:4436).T-cell receptors and costimulatory molecules are required for activationof T-cells and in development of inflammation driven lung fibrosis(Elhai M, Avouac J, Hoffmann-Vold A, Ruzehaji N, Amiar O, Ruiz B,Brahiti H, Ponsoye M, Fréchet M, Burgevin A, Pezet S, Sadoine J,Guilbert T, Nicco C, Akiba H, Heissmeyer V, Subramaniam A, Resnick R,Molberg O, Kahan A, Chiocchia G, Allanore Y (2016). Proc Natl Acad SciUSA 113:E3901-10). It was found that downregulation of T cell receptorcomplex genes (CD3E, CD3G, CD4, and CD8A) had the highest associationsin pathway analyses. MIA-602 may thus play an important role in lungtissue by modification of T-cell signaling and potentially reducinginflammation and fibrosis.

These data show that GHRH-R is present in human lungs; and, in arelevant in vivo model, lung fibrosis is modulated by its inhibition.Functional findings implicate GHRH and GH in fibrosing lung disease, andthey are consistent with demonstrated effects of the GHRH-R antagoniston mitochondrial respiration and fibroblast cytotoxicity. MIA-602inhibits intracellular signaling pathways, including p21 activatedkinase/STAT3/NFκB and PI3K/AKT, in addition to having intrinsicantioxidant activity. Further, it could support mitochondrial functionand maintain autophagy, minimizing fibrosis.

1. A peptide comprising Formula I: (SEQ ID NO: 1)X0-Tyr-DArg-Asp-Ala-Ile-X6-Thr-X8-X9-X10-X11-X12-Val-Leu-Abu-Gln-Leu-Ser-Ala-X20-X21-Leu-Leu-Gln-Asp-Ile-Nle-DArg-X29-X30,

wherein X0 is 5FPhAC-Ada, p-cePhAC, D-Phe-Ada, or PhAC-Ada; X6 is 5FPheor Cpa; X8 is Ala or Asn; X9 is Arg or Har; X10 is Tyr(Me), Amp or5FPhe; X11 is Arg or His; X12 is Lys or Orn; X20 is Arg or His; X21 isLys or Orn; X29 is Har, Har-NH2 or Har-NHCH3; and X30 is present orabsent and, when present, is Ada-NH2, Ada-NHCH or Ada-NHCH2CH3, or apharmaceutically acceptable salt thereof.
 2. The peptide of claim 1,comprising the amino acid sequence of Formula II: (SEQ ID NO: 2)5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-X6-Thr-X8-Har-X10-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-X29-X30,

wherein X6 is 5FPhe or Cpa, X8 is Ala or Asn, X10 is Tyr(Me) or 5FPhe,X29 is Har or Har-NHCH3, and X30 is present or absent and, when present,is Ada-NH2 or Ada-NHCH3, or a pharmaceutically acceptable salt thereof.3. The peptide of claim 2, comprising the amino acid sequence: (a) (AVR-235, SEQ ID NO: 3) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-NHCH3; (b)(AVR-333, SEQ ID NO: 4) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har- Ada-NH2; (c) (AVR-352, SEQ ID NO: 5) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har- Ada-NH2; (d) (AVR-353, SEQ ID NO: 6) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada- NH2; or (e) (AVR-354, SEQ ID NO: 7) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH3.


4. The peptide of claim 1, comprising the amino acid sequence: (a) (AVR-235, SEQ ID NO: 3) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-NHCH3; (b) (AVR-333, SEQ ID NO: 4) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har- Ada-NH2; (c) (AVR-352, SEQ ID NO: 5) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har- Ada-NH2; (d) (AVR-353, SEQ ID NO: 6) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada- NH2; (e) (AVR-354, SEQ ID NO: 7) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH3; (f) (AVR-104, SEQ ID NO: 8) PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH2; (g) (AVR-107, SEQ ID NO: 9) PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH2; (h)  (AVR-116, SEQ ID NO: 10)PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-5FPhe-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH2; (i)  (AVR-120, SEQ ID NO: 11)D-Phe-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NH2; (j) (AVR-201, SEQ ID NO: 12) PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Arg-Amp-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH3; (j) (AVR-234, SEQ ID NO: 13) PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH3; (k) (AVR-321, SEQ ID NO: 14) PhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Aoc- NHCH3; (l) (AVR-322, SEQ ID NO: 15) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Har-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har- Aoc-NHCH3; (m) (AVR-542, SEQ ID NO: 16) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-NHCH3; (n) (AVR-543, SEQ ID NO: 17) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada- NHCH3; (o) (AVR-552, SEQ ID NO: 18) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Ala-Har-5FPhe-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har-Ada- NH2; (p) (AVR-553, SEQ ID NO: 19) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-5FPhe-Thr-Ala-Har-Tyr(Me)-His-Orn-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har- Ada-NH2; or (q) (AVR-620, SEQ ID NO: 20) 5FPhAC-Ada-Tyr-DArg-Asp-Ala-Ile-Cpa-Thr-Asn-Arg-Tyr(Me)-Arg-Lys-Val-Leu-Abu-Gln-Leu-Ser-Ala-His-Orn-Leu-Leu-Gln-Asp-Ile-Nle-DArg-Har- Ada-NH2.


5. A method of inhibiting tumor growth, the method comprisingadministering to a subject an effective amount of the peptide ofclaim
 1. 6. The method of claim 5, wherein the tumor is prostate cancer,breast cancer, lung cancer, colorectal cancer, melanoma, bladder cancer,brain/CNS cancer, cervical cancer, esophageal cancer, stomach cancer,colon cancer, head/neck cancer, kidney cancer, liver cancer, lymphoma,ovarian cancer, pancreatic cancer, thyroid cancer, glioblastoma,leukemia or sarcoma.
 7. The method of claim 5, wherein the tumor is aprimary tumor or a metastatic tumor.
 8. A method of treating cancer, themethod comprising administering to a subject an effective amount of thepeptide of claim
 1. 9. The method of claim 8, wherein the cancer isprostate cancer, breast cancer, lung cancer, colorectal cancer,melanoma, bladder cancer, brain/CNS cancer, cervical cancer, esophagealcancer, stomach cancer, colon cancer, head/neck cancer, kidney cancer,liver cancer, lymphoma, ovarian cancer, pancreatic cancer, thyroidcancer, glioblastoma, leukemia or sarcoma. 10.-30. (canceled)
 31. Themethod of claim 5, wherein the subject is identified as being in need oftreatment before the administration step.
 32. The method of claim 5,wherein the therapeutically effective amount of the GHRH peptide isadministered intravenously, subcutaneously, intratracheally or orally.33.-35. (canceled)